ESI-CID spectral characterization and differentiation of the cross links of thymine formed by one electron oxidation with SO4 ●-

Article abstract of DOI:10.1016/j.ijms.2019.05.001

The analysis of urinary nucleosides is becoming a very good tool in the diagnosis of diseases like AIDS and cancer and consequently the identification of modified nucleosides using mass spectrometry is an area of utmost importance. In this context, there is a high relevance in the understanding of the mechanism of collisionally induced dissociation (CID) of these heterocyclics. The present work characterizes and differentiates the cross-linked products formed by one electron oxidation of thymine (T). Three dimers A, B and C, were analyzed, out of which the dimer A is a C5–C5’ cross link of two T molecules containing a fused tetrahydrofuran ring, while B and C are N–C cross linked products. B and C showed very similar fragmentation pattern but that of A was different. The differentiation between these three were made by monitoring two characteristic peaks, the water loss peak ([M+H]⁺) and the protonated T fragment ([T + H]⁺). The probable mechanism of formation of these fragments and their CID mechanism in both positive and negative ionization modes are also explained. The neutral losses of NH₃, H₂O and NHCO were the prominent mechanism in the positive mode, while in the negative mode, only NHCO and CO losses were observed.

Full text of DOI:10.1016/j.ijms.2019.05.001

Accepted Manuscript
ESI-CID spectral characterization and differentiation of the cross links of thymine
●-
formed by one electron oxidation with SO
4
Jisha Chandran, N.R. Vishnu, Usha K. Aravind, C.T. Aravindakumar
PII:
S1387-3806(18)30492-5
DOI:
https://doi.org/10.1016/j.ijms.2019.05.001
MASPEC 16147
Reference:
To appear in: International Journal of Mass Spectrometry
Received Date: 5 December 2018
Revised Date: 17 April 2019
Accepted Date: 2 May 2019
Please cite this article as: J. Chandran, N.R. Vishnu, U.K. Aravind, C.T. Aravindakumar, ESI-CID
spectral characterization and differentiation of the cross links of thymine formed by one electron
●-
oxidation with SO , International Journal of Mass Spectrometry (2019), doi: https://doi.org/10.1016/
4
j.ijms.2019.05.001.
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ESI-CID spectral characterization and differentiation of the cross links of
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thymine formed by one electron oxidation with SO4
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Jisha Chandrana, Vishnu N. R.b, Usha K. Aravindc and C. T. Aravindakumara,b
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aInter University Instrumentation Center, bSchool of Environmental Sciences, cAdvanced Centre
of Environmental Studies and Sustainable Development, Mahatma Gandhi University,
Kottayam, Kerala, India
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Abstract
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The analysis of urinary nucleosides is becoming a very good tool in the diagnosis of diseases like
AIDS and cancer and consequently the identification of modified nucleosides using mass
spectrometry is an area of utmost importance. In this context, there is a high relevance in the
understanding of the mechanism of collisionally induced dissociation (CID) of these
heterocyclics. The present work characterizes and differentiates the cross-linked products formed
by one electron oxidation of thymine (T). Three dimers A, B and C, were analyzed, out of which
the dimer A is a C5-C5’ cross link of two T molecules containing a fused tetrahydrofuran ring,
while B and C are N-C cross linked products. B and C showed very similar fragmentation
pattern but that of A was different. The differentiation between these three were made by
monitoring two characteristic peaks, the water loss peak ([M+H]+) and the protonated T fragment
([T+H]+). The probable mechanism of formation of these fragments and their CID mechanism in
both positive and negative ionization modes are also explained. The neutral losses of NH3, H2O
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and NHCO were the prominent mechanism in the positive mode, while in the negative mode,
only NHCO and CO losses were observed.
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Keywords: DNA, thymine cross-links, ESI-CID, one electron oxidation, fragmentation
mechanism, neutral loss
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1. Introduction
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Analysis of modified nucleic acid constituents is an area of tremendous research interest as it
now plays a major role in the diagnosis of many diseases like aids and cancer [1]. There are as
many as 100 oxidatively modified lesions reported in isolated DNA and model compounds till
now under various oxidative stress conditions [2]. The fact that formation of new modified
products different from those formed at the constituent (nucleosides and nucleotides) level is
being reported at the level of DNA [3] makes this an area of sustained research interest. Among
the various agents causing oxidative damage to DNA, ionizing radiation is one of the important
and most investigated one [2, 4-13]. It imparts its effects directly and indirectly on DNA and
RNA. In direct effect, the photons are directly absorbed by the DNA (or RNA) molecule
resulting in electronically excited state and further decompose to radical cations and electrons.
However, in indirect effect, the reactive radicals formed from water radiolysis including
hydroxyl radical (●OH) reacts with DNA (or RNA) giving rise to other organic radicals. These
radicals and radical cations react with the water in the surrounding medium to form stable
oxidative modifications. When the radical cations are generated in double helical DNA, the
charge can migrate to other parts by a long range tunneling (super exchange), or by hopping
mechanism ultimately resulting in DNA damage [14]. For modeling such reactions at the
constituent level, the radical cations are generated by using a variety of one electron oxidants
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such as SO4•- [15, 16] and Br2•- [17, 18], direct photoionization by the absorption of two photons
[19], and triplet sensitized electron transfer to excited carbonyl compounds [20, 21].
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With the advent of highly sophisticated analytical tools, the detection and identification of new
photoproducts or radical mediated products have become more precise resulting in a faster and
efficient elucidation of novel mechanistic pathways in the field of DNA free radical chemistry
[8]. The UPLC-ESI-CID (ultra-high-performance liquid chromatography- electrospray
ionization- collisionally induced dissociation) technique is one such technique with high
specificity and sensitivity, which led to the development of strategies for identification of
specific modifications applicable to clinical diagnostic purposes [8, 22]. The ESI-CID analysis of
urine, for the detection and identification of modified nucleosides and adducts formed by
genotoxic chemicals with DNA, has now become a routine process in the diagnosis of cancer and
aids [23]. All these point out to the prevailing interest in the development of ESI-CID based
identification and characterization strategies for nucleosides, modified nucleosides as well as
DNA adducts. A thorough understanding of the ESI-CID of the DNA constituents and their
modified forms is necessary to reach this goal. But the major challenge is that ESI-CID reactions
of the ions of these heterocyclic compounds are complex processes to be explained. The CID
mechanism of adenine [24], guanine [25] and uracil [26, 27] and of their corresponding
nucleosides [28] is documented with the aid of isotopic labeling studies and theoretical
calculations. This can be extended to modified nucleosides, photoproducts and cross linked
products adding to the wealth of knowledge on the CID mechanisms of protonated and
deprotonated DNA constituents and their modified forms.
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Among the oxidative modifications of DNA and/or its constituents, the dimeric structures play a
major role as they are often formed as a result of radical recombination reactions under hypoxic
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conditions [29-31]. There are reports on the formation of DNA-DNA cross links [32, 33] in cell
lines also and the photodimers are found to be the most toxic among them [34, 35]. The
biological implication of the formation of these cross links makes the ESI-CID studies on these
molecules relevant. It is to be noted that, in model systems like thymine (T) in aqueous solution,
different cross links with same elemental composition are formed by covalent bond formation
between different sites on the T moieties. Previously, Hatta et al have reported the formation of
N(1)-C(5’) and N(1)-C(6’) linked dimer hydrates of 5-substituted uracils by electrochemical and
2-methyl-1,4-naphthoquinone photosensitized oxidation reactions in aqueous medium. In the
case of T, N(1)-C(5’) and N(1)-C(6’) dimer hydrates along with two novel stereo isomeric C(5)-
C(5’) linked dimers were reported to be formed [36]. There is also another report on the
formation of a number of dimer and trimer hydrates on UV irradiation of the aqueous solution of
T [37] in addition to the photodimers.
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In the present paper, we extend the ESI-MSMS studies to three isomeric T cross links formed by
the one electron oxidation reactions. Previous studies on dimeric structures (photodimers) using
mass spectrometry were focused on recognizing certain representative peaks in the spectra for
their easy detection and identification [38]. But here a detailed ESI-CID study of the cross links
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of T formed by one electron oxidation with SO4 in aqueous solution is presented and is
compared with that of the photoproducts. Out of the three cross linked structures, two are C-N
cross links and one is a C-C cross link. The various logical neutral losses from these cross links
were carefully examined and tabulated both in the positive and the negative modes. It is to be
remembered that a slight modification on these types of structures may completely alter the
fragmentation pattern [27]. Taking two peaks in the CID spectra corresponding to the protonated
T fragment and the water loss fragment as the indicator peaks, an attempt to distinguish between
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the isomeric cross links has been made. The complexity of the ion processes and the biological
relevance of the cross links makes the study interesting in an analytical point of view.
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2. Materials and methods
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Thymine (T) was purchased from Sigma Aldrich and K2S2O8 from SRL. All the solutions were
prepared with water purified by CascadaTM Lab Water Systems and of resistivity 18.2 MΩ.cm.
The sulfate radicals were produced by the UV-photolysis of peroxide as reported elsewhere [39].
The solution containing T (10-4 M) and K2S2O8 (10-3 M) was subjected to UV irradiation at a
wavelength of 254 nm for about 2 minutes. The solution was then directly subjected to UPLC-
ESI-CID analysis.
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The various products formed by the SO4•- mediated transformation of T were analysed by Waters
Xevo G2 QToF with Acquity H class UPLC, Aquity TUV detector and ESI source. The UPLC
separation was carried out with BEH C18 (50 mm×2.1 mm×1.7µm) column. The instrument was
first tuned with the parent compound, for the maximum intensity in both positive and negative
modes. The ionization conditions in the positive mode were as follows: capillary voltage: 2.5 kV,
sampling cone voltage: 35 V and extraction cone voltage: 3.5 V. Nitrogen was used as the
desolvation and nebulizing gas at a flow rate of 50 and 900 L/hr, respectively. The products were
eluted isocratically with water (0.1% formic acid) and methanol (95:5) and the spectra were
recorded both in positive and negative modes of ionization.
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The energy resolved mass spectrometric analyses (ERMS) were carried out to get an idea on the
evolution of the various fragments with increase in collision energy. The ERMS curves are the
plots of the relative ion current for that particular fragment against collision energy. The relative
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ion currents were calculated using the following equation [40] were relC is the relative ion
current, Ci and Cp are that of the specific ion of interest and the parent ion current respectively.
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relC =
× 100
3. Results and Discussion
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The three isomeric cross links of T described in the present study were produced by SO4 from
the UV photolysis of K2S2O8. The experiments were conducted in the native pH and repeated at
pH 7 for confirming the identity of the dimers with that reported by Hatta et al in the anodic
oxidation of T. The solution after photolysis was subjected to UPLC-ESI-CID analysis and all
the products were analyzed. In addition to the three cross linked products (A, B and C),
monomeric oxidation products were also observed. Thymine glycols, 5-hydroxymethyl thymine
and 5-formyl uracil were the major monomeric products obtained. The dimers are having the
same m/z (m/z = 269.0888 in positive mode and m/z = 267.0730 in negative mode) and
subsequently same elemental composition (C10H13N4O5 in positive mode and C10H11N4O5 in
negative mode). The total ion chromatogram obtained for A, B and C is given in the fig. 1. The
collisionally induced dissociation (CID) spectra of these compounds, in the positive and negative
modes are given in the fig. 2 and 3 respectively. Four cross linked structures have been reported
in anodic oxidation: meso and racemic mixture of C(5)-C(5’) cross links with a fused
tetrahydrofuran structure, N(1)-C(5’) and N(1)-C(6’) cross linked dimers [36]. But in the present
case, only three were observed. The formation of the N(1)-C(5’) and N(1)-C(6’) cross linked
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dimers from the reaction of T with strongly oxidizing SO4 (at pH 7) is also known [36]. The
presence of peak at m/z 127 (corresponding to the [T+H]+ fragment) in B and C indicated the
presence of C-N cross link. However, no fragment corresponding to [T+H]+ or [M+H-T]+ was
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observed in the CID spectrum of A, ruling out the possibility of a C-N cross link. A stable C-C
cross link with the elemental composition C10H12N4O5 could most probably have a structure
corresponding to that of C(5)-C(5’) cross link with a fused tetrahydrofuran ring. A, B and C are
assigned as the C(5)-C(5’) dimer with a fused tetrahydrofuran structure, N(1)-C(5’) and N(1)-
C(6’) cross linked dimers respectively comparing with the peak elution pattern from the
reversed phase C18 HPLC column as reported by Hatta et al [36]. The structures are given in
the fig. 4. No structural information on whether the C(5)-C(5’) dimer is meso or racemic form
was obtained from the ESI-CID analysis, but from the order of elution from the same column it
is tentatively assigned as the meso form. Further evidence for the structural assignment of B and
C are provided on comparison with the T photodimers. In addition to that, dimers B and C are
structurally differentiated using the [M+H-H2O]+ and [T+H]+ fragments.
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Fig.1. The total ion chromatogram of the dimers in the positive mode, collision energy: 15 eV.
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The formation of N(1)-C(5’) and N(1)-C(6’) cross linked dimers has been proposed through a
N(1)-centered radical intermediate while the C(5)-C(5’) cross link can be formed via a C(5)
centered radical [36]. The structures were further confirmed by considering the mechanism of
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one electron oxidation of T under the persulfate-UV photolysis. The SO4 , produced as a result
of homolysis of S2O82- on UV irradiation, is a powerful one electron oxidant which is often used
in modeling the direct effect of the ionizing radiation on DNA bases [15]. The EPR and pulse
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radiolysis studies on the reactions of sulfate radical anion (SO4 ) with the purines and
pyrimidines reveal that the reaction is initiated by a stable adduct formation which subsequently
decomposes to radical cations and SO42- [16, 41-43]. The stabilization of the radical cation takes
place predominantly through two ways: either via deprotonation at the N(1) or N(3) or by the
formation of –OH adduct radical through the nucleophilic addition of H2O [41]. In a study of
methylated pyrimidines with SO4•- using pulse radiolysis, Lomoth et al has reported that initially
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a SO4•- adduct is formed which decays to a radical cation followed by deprotonation at N(1) [41].
All these show that the N(1) centered radical is the species formed in highest concentration
during the process, which supports the formation of the C-N linked dimers. The presence of
C(5)-centered radicals were indicated by the presence of glycols, 5-hydroxymethyl and 5-formyl
uracil [41, 44] in the present case, which in turn supports the formation of the C(5)-C(5’) cross
link.
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Fig.2. CID spectra of the dimers A, B and C in the positive mode, collision energy: 15eV
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Fig.3. CID spectra of the dimers A, B and C in the negative mode, collision energy: 15 eV
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Fig.4. The structures of the cross-linked products formed by the one electron oxidation of T.
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The ESI-MS/MS spectra were acquired both in the positive and negative modes. In the negative
mode all the dimers showed the same fragmentation pattern with only difference being in the
relative intensity of peaks as well as the absence of the deprotonated T fragment in the spectrum
of A. However, in the positive mode the CID spectrum of A was different from that of B and C.
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The CID spectra of B and C were found to contain same peaks, differing only in intensity.
Elemental compositions of both the parent ion and the fragments are deduced from the accurate
mass. The logical neutral losses were established both in the positive and negative modes which
are summarized in the table S1 and S2 (Supporting Information). The data are compared with the
ESI-MS/MS fragmentation data of far UV induced photo products of T available in literature.
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The structures of the dimers are quite complicated with 9 sites of protonation and 4 sites of
deprotonation. The compounds decompose by strictly following the even electron rule as
reported in the case of the monomer, uracil [26]. All the fragments observed could be explained
on the basis of neutral losses. The NHCO and CO losses were commonly observed in the
positive as well as negative mode. In the positive mode, NH3 and H2O losses were additionally
observed. The spectra in the negative mode were simpler as the major peaks were found to be
arising from sequential elimination of NHCO and CO. At collision energy of 14 eV (collision
energy at which the intensities of the parent ions were comparable to that of the corresponding
dimers in the positive mode), [M-H-NHCO]- was found to be the base peak for dimer A. But for
B and C, [M-H-NHCO-CO]- is the base peak. Peaks corresponding to the loss of 2 to 3
molecules of NHCO are also observed in the spectrum in the negative mode. The protonated
forms of dimers B and C sequentially lose H2O, NH3, NHCO, NH3 and CO along with the
formation of a protonated T fragment ([T+H]+). For dimer A, there is a sequential loss of NHCO,
NH3 and CO. The CID spectra of the deprotonated dimers are dominated by [M-H-NHCO]- and
[M-H-CO]- peaks. It is interesting to note that, a peak at m/z 181.06 (C8H9N2O3) is observed in
both positive and negative modes. By analyzing the elemental composition, it was found that its
origin is from the sequential loss of NHCO, NH3 and CO in the positive mode. But from the
deprotonated dimers, it can be formed by the loss of two NHCO molecules.
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The ERMS break down curves of the protonated parent molecules are shown in the fig. 5a, from
which a relative stability towards CID is predicted. Protonated B was found to be much more
stable than C. The normalized abundances of all the product ions are plotted against collision
energy for the similar structures B and C and are given in fig.5b and 5c. Even though the peaks
are similar, there is striking difference in their abundance of formation from the protonated
dimers B and C, which is evident from the figures. The fragment with the highest value for the
relative ion current was found to be m/z 251.07 ([M+H-H2O]+) in the case of B and m/z 181.06
([M+H-(NHCO)-NH3-CO]+) for C. The [M+H-(NHCO)-NH3-CO]+ fragment is formed from C
even at very low collision energy and the abundance increases with collision energy. No
fragments were observed which could be arising from the [M+H-H2O]+ peaks.
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Fig.5. (a) The plot of relative ion current vs. collision energy for B and C; the variation of
evolution of fragments with collision energy from (b) B and (c) C, (d) relative ion current vs.
collision energy for the deprotonated B, C and the fragments; the closed symbols represent
deprotonated B and its fragments and open symbols deprotonated C and corresponding
fragments.
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The CID of protonated A differs from that of B and C because of the absence of [M+H-H2O]+,
[M+H-NH3]+ and [T+H]+. The protonated form of dimer A ([A+H]+), which doesn’t possess a
free OH, undergoes a loss of NHCO to give a fragment at m/z 226, the relative abundance being
22%. But in the case of [B+H]+ and [C+H]+, the relative abundance of [M+H-NHCO]+ was very
less, 3% and 1%, respectively. Since [M+H-NHCO-NH3]+ and [M+H-NHCO-NH3-CO]+ are of
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sufficient intensity in this case, it can be concluded that [M+H-NHCO]+ is unstable and easily
decomposes to stabilized structures by losing NH3 and CO consecutively. It is well established in
the case of uracil that only those ions which are extensively stabilized by resonance are present
in the mass spectrum and the ions with localized charges are unstable and of low abundance [26].
Substitution at the ring clearly affects the fragmentation mechanism by altering the site and
extent of protonation, sometimes resulting in increased dissociation reactions and charge
retention at the site of substitution [27]. Dimer A can be visualized as uracil substituted on both
C-5 and C-6. There are already reports that the C-5 substituted 2-thiouracil exhibits lesser
intensity of the ammonia loss fragment than the parent 2-thiouracil [27]. The same mechanism
may operate here also, suppressing the loss of ammonia. Previous studies report the loss of
NHCO from pyrimidines [26, 27] and it is proposed that the elimination of NHCO is the favored
mechanism in dimer A.
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Comparing these ESI-CID spectra of these cross links with that of photodimers, it was found that
the spectra of the latter were simple compared to the former. For the 6-4 photoproduct and the
spore photoproduct, the spectra consisted of [M+H-NHCO]+ and the [T+H]+ fragments [38]. The
6-4 photoproduct gives an H2O loss peak while there is an NH3 loss peak for the spore
photoproduct. For the cyclobutane dimers also, the major fragments observed in the positive
mode are the [M+H-H2O]+, [M+H-NHCO]+ and [T+H]+. The peaks [M+H-(NHCO)-NH3]+ or
[M+H-(NHCO)-NH3-CO]+ were not observed in the case of the photodimers [38].
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From the table S1 (Supporting Information), it is clear that the relative propensity of water loss is
greater for [B+H]+ than [C+H]+, even though it occurs in both the cases. At a collision energy of
16 eV, the base peak is the [M+H-H2O]+ in the case of [B+H]+ while its relative abundance is
only 14% for [C+H]+. The dependence of the emergence of the [M+H-H2O]+ peak on the
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collision energy was examined by performing the ERMS analyses. The CID experiment was
repeated at a collision energy starting from 4 to 22 kV, and it is found that the water loss is
highly favorable for B at all these collision energies (fig.6). The water loss peak is not formed
from dimer A. It was observed that [M+H-H2O]+ occurs as the major fragment in the (6-4)
photoproduct and its corresponding Dewar valence isomer also. Both contain a free OH group
[38]. It is to be noted that the other photoproducts, the cyclobutane thymine dimer and the spore
photoproduct, didn’t undergo water loss to produce the corresponding ion of sufficient intensity
[38]. It is proposed that the free -OH group is the major site of protonation for these compounds
in the positive mode. A mechanism of water loss and stabilization of the [M+H-H2O]+ ion is
proposed which explains the difference in the formation of the water loss fragment from B and
C. The mechanism is shown in Scheme 2.
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Fig.6. The formation of the [M+H-H2O]+ from the protonated dimers [A+H]+, [B+H]+ and
[C+H]+
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The [A+H-H2O]+ and [B+H-H2O]+ ions formed in the case of B and C are proposed to be
stabilized by the resonance as shown in the scheme 1. The diketo form is more stable in the
solution state, but a tautomerization to dienol form is more feasible in the gaseous phase [27].
Hence a resonance stabilization arising from the dienol form is proposed for the [B+H-H2O]+ ion.
A reaction is energetically favored if the resulting species is highly stabilized. For C, such
resonance stabilization is hindered as the positive charge must reside on nitrogen atom which is
unlikely. But the ion [C+H-H2O]+ is stable to some extent as it is a tertiary carbocation which
can be the driving force for the water loss. The [M+H-H2O]+ peak from the 6-4 photoproduct
was found to be of low intensity and in that case also, the resulting ion is similar to [C+H-
H2O]+[38].
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Scheme 1. Stabilization mechanism of the dehydrated [M+H]+ of B and C.
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The formation of [T+H]+ is observed in the CID of B and C but not in the case of A. The
fragment is observed in the spore photoproduct, (6-4) as well as the cis-anti and trans-anti forms
of cyclobutane thymine dimers but not observed for cis-syn and trans-syn forms of cyclobutane
thymine dimers [38]. The dimeric structures B and C are similar in structure to thymidine where
there is an N-C bond. Also, there is an -OH group in close proximity a proton transfer to occur.
The possibility of occurrence of the protonated thymine fragment, which require the breakage of
the cross link, is feasible for B and C but not for A. The evolution of the [T+H]+ from the
protonated dimers are represented in fig.7. The process requires the cleavage of a C-N bond for
B and C, whereas in the case of A, the cleavage of a tetrahydrofuran ring should occur which is
much less feasible. A possible mechanism of the formation of [T+H]+ (scheme 2) is proposed to
follow the same pathway in the case of nucleosides [45]. Protonation takes place at the ring 1,
followed by an intra-molecular proton transfer from the OH group in the close proximity
resulting in the heterocyclic cleavage of the N-glycosidic bond. From the ERMS data, it is clear
that this peak emerges slowly with collision energy. There is no significant difference in the
formation of [T+H]+ from B and C except at higher collision energies. At higher collision
energies, B shows slightly higher propensity of formation of [T+H]+ which is likely due to the
steric hindrance caused by the C5’-methyl group.
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Fig. 7. The formation of the [T+H]+ from the protonated dimers A, B and C.
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Scheme 2. The possible mechanism of formation of [T+H]+ from [B+H]+ and [C+H]+.
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Scheme 3. The mechanism of formation of [T-H]- from the [B-H]- and [C-H]-.
In most of the studies on the gas phase fragmentation of pyrimidines, the mechanism of the
chemistry of only the protonated molecule is explained [26, 27]. The details of the gas phase
chemistry of deprotonated nucleosides are very less in literature. The present study gives light
into the mechanism of fragmentation of the thymine cross links in the negative mode also. By a
close observation, it was found that the spectra are very simple and the peaks correspond to the
NHCO and CO losses from the precursor. For dimer A, the peak corresponding to the fragment
[M-H-NHCO]- was found to be the base peak but for the dimers B and C, the intensity of that
peak was found to be less (5% and 3% respectively). It may decompose by losing a CO molecule
to form the base peak at m/z 196.07 ([M-H-NHCO-CO]-). The loss of two NHCO (peak with m/z
181.06) molecules is also highly favored for all the three dimers. A minor process, a CO loss is
also observed from the [M-H-2NHCO]-. A peak at m/z 138.0001 was observed in the case of A,
but that was of very less intensity in the case of B and C. From the elemental composition, it was
found to be [M-H-3NHCO]-. A deprotonated thymine fragment was found to be arising from B
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and C. The mechanism of formation of the deprotonated thymine is given in the scheme 5. The
breakdown curves of deprotonated B and C were found to be coinciding indicating they are equal
in stability. A similar situation is encountered for the evolution curves of the product ions also
(fig.5d). The ERMS data substantiates the formation of the fragments also. The fragment [M-H-
NHCO]- is formed even at low collision energy and when its relative ion current reaches around
40%, other fragments begin to show up.
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4. Conclusion
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The characterization of the dimeric structures formed by the one electron oxidation of T was
made under ESI-MSMS condition. There are many reports on CID mechanism of the nitrogen
bases but detailed studies on the cross links are rare. Only certain peaks were characterized in
order to assess the possibility of being used as the marker peak for identification in the real
samples [38]. The present study reports and accounts for almost all the peaks formed in the
spectra in the positive and negative mode. The spectra in the negative mode are characterized by
the losses of CO and NHCO. A mechanism of formation and stabilization of the [M+H-H2O]+
and [T+H]+ ions is proposed. Only C-N cross linked dimers has been reported in the one electron
oxidation with SO4•- previously, and in the present work evidence for the formation of dimeric
structure with fused tetrahydrofuran ring is also provided. In effect, the present study serves the
purpose of unraveling the gas phase fragmentation chemistry of the structurally complex and
biologically relevant heterocyclic compounds, T cross links.
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Acknowledgement
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JC is thankful to UGC for the financial support under the JRF scheme. Part of the financial
support for this work was from KSCSTE, Thiruvananthapuram
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Captions of figures and schemes
Fig.1. The total ion chromatogram of the dimers in the positive mode, collision energy: 15 eV.
Fig.2. CID spectra of the dimers A, B and C in the positive mode, collision energy: 15eV
Fig.3. CID spectra of the dimers A, B and C in the negative mode, collision energy: 14 eV
Fig.4. The structures of the cross-linked products formed by the one electron oxidation of T.
Fig.5. (a) The plot of relative ion current vs. collision energy for B and C; the variation of
evolution of fragments with collision energy from (b) B and (c) C, (d) relative ion current vs.
collision energy for the deprotonated B, C and the fragments; the closed symbols represent
deprotonated B and its fragments and open symbols deprotonated C and corresponding
fragments.
Fig.6. The formation of the [M+H-H2O]+ from the protonated dimers [A+H]+, [B+H]+ and
[C+H]+
Fig. 7. The formation of the [T+H]+ from the protonated dimers A, B and C.
Scheme 1. Stabilization mechanism of the dehydrated [M+H]+ of B and C.
Scheme 2. The possible mechanism of formation of [T+H]+ from [B+H]+ and [C+H]+.
Scheme 3. The mechanism of formation of [T-H]- from the [B-H]- and [C-H]-.

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