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Methods

Experimental

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    The experimental
data is obtained using collision induced dissociation (CID)in a LCQ Deca plus
XP ion trap mass spectrometer. Dilute samples (10-4 M) of the methyladenine
species are prepared in a 1:1 distilled water:methanol solution (HPLC grade H2O,
Sigma Aldrich, HPLC grade methanol, Fischer Scientific).  1, 3, and 7- methyladenine (98% Sigma Aldrich 1-methyladenine,
Sigma Aldrich 3-methyladenine autophagy inhibitor, 97% Fischer Scientific
7-methyladenine) are deprotonated by electrospray ionization (ESI). The
experimental layout of the ESI process is shown in Figure 2 and the expected
structures are displayed in Figure 1. A potential difference of 4.5 kV is
applied between the spray nozzle and the heated ion transfer tube (250 °C)
where the molecules are de-solvated, and a sample flow rate is maintained at 15
?L min–1. The deprotonated methyladenine ions travel from the ESI
source and are focused by an octapole and quadrupole into the ion trap (Figure
4) where the ions then collide with helium at a pressure of 0.177 Torr.
This pressure is determined through a calibration process described in our
previous work.11 Tandem mass spectrometry (MSn) is used
in order to isolate a desired ion and conduct additional fragmentation. The
normalized collision energy (NCE) is varied up to 50% in order to fully
fragment a specific ion within the trap. In addition, the activation quotient
and activation times are set to .250 and 30 ms, respectively.

 

 

 

Figure 2.  The
schematic shown above describes the experimental design of the electrospray
ionization process. 12 The schematic shows a Taylor cone containing
the anions being drawn into the heated ion transfer tube. From the ion transfer
tube molecules are de-solvated and focused through a quadrupole and octapole
into the ion trap.

 

 

 

 

 

 

Computational

    Gaussian 09 software is utilized to further analyze the fragments of the
methyladenine species produced by CID. Density functional theory is also employed
in conjunction with Gaussian 09 at the B3LYP/6-311++G(d,p) level of
theory. Potential energy surfaces (PES) and acidity calculations are obtained
by optimization of molecular geometries alongside frequency calculations of the
local minima and maxima of the PES, respectively. Gas-phase acidities are
calculated and reported as the change in the Gibbs free energy for reaction at
298K.  Associated zero point energy
corrections are applied.

 

Figure 3. The staggered
spectra above displays the unique experimental spectrum for each of the
methyladenine compounds. Shown in red, is the deprotonated anionic form (parent
ion) of the functionalized purine molecule occurring at m/z 148 for all the
methyladenine species. In all of the above spectra, a peak at m/z 133 was
detected. This peak is characteristic of a commonly seen loss of methyl
radical. In addition to the peaks at m/z 133, 1-methyladenine, shown in black,
displays a unique peak at m/z 107 likely resulting from the loss of CH3CN.
Seen in blue, the 7-methyladenine also displays a unique peak, this time
appearing at m/z 121. This peak is indicative of a loss of HCN. For all of the above spectra with the
exception of the parent ion peak a normalized collision energy of 45% was
applied, with an activation quotient and time of .250 and 30 ms, respectively.

 

Results And Discussion

     Herein, we
report the gas phase acidities, spectral analysis and density functional theory
(DFT) computations regarding collision induced dissociation of 1, 3, and
7-methyladenine within the LCQ Deca plus XP Quadrupole ion trap mass
spectrometer. Determined computationally, the gas phase acidities of the purine
species led to the proposed anionic structure (Figure 1). These geometry optimizations
described the most stable structures to be 1, and 7-methyladenine deprotonated
at their N9 position while 3-methyladenine experiences effective deprotonation
at it N13 site. It is important to note that acidity calculations were
performed for all potential sites including C-H bonds. Although deprotonation
of the C-H bond is possible upon ESI,
in the differing species, the occurrence of C-H deprotonation led to relatively
high energy conjugate bases. Upon collision induced dissociation within the ion
trap (Figure 4) each of the methyladenine compounds displayed unique spectral
differences for each of the anionic molecules produced (Figure 3). In red is
the distinct peak corresponding to the “parent ion”, or deprotonated methyladenine
compound, occurring at m/z 148
in accordance with the mass of each ion. While each
spectrum is unique, similarities are identifiable in each. Primarily, each
species of ion displays a peak at m/z 133. This peak corresponds to a loss of
15 AMUs and can most reasonably be explained by the loss of the methyl
substituent in its radical form. In addition to the peaks at m/z 133,
1-methyladenine, shown in black, displays a unique peak at m/z 107. This peak
represents a loss of 41 AMUs and likely results from the fragmentation of CH3CN.
Seen in blue, the 7-methyladenine also displays a unique peak appearing at m/z
121. The mass of the fragment represented by this peak is likely indicative of
the parent ion having a lost an HCN molecule. It is considered highly likely
that the unique losses may be isomers of CH3CN and HCN seen in
previous literature.12,14,15 All of the spectra were obtained at an
unusually low normalized collision energy value (45%). Cole et al2 describes
complete dissociation to occur well over an NCE value of 50 percent. This
anomaly is likely to be caused by the high coefficient of pressure (.177 torr) within
the ion trap, thus causing more frequent and higher energy collisions.  

 

Figure 4.  The
schematic above describes of the focusing of ions by a quadrupole and octapole
into the ion trap. Once inside the trap, the ions undergo collision induced
dissociation with helium gas. This process results in anionic fragments that
can then be detected by an electron multiplier.

    

     Computations revealed the potential energy surfaces for the loss of methyl
radical for each of the methyladenine molecules (Figure 5). The dissociation
events are considered to take place without a transition state and occur at
unusually high energys. For 1-methyladenine the energy of the resultant
fragments lies at approximately 92.79 kcal·mol-1 whereas
3-methyladenine exhibited a slighly lower energy of 91.2 kcal·mol-1. The lowest energy severance is shown in 7-methyladenine
around 87.67 kcal ·mol-1. Excited state intermolecular proton
tranfers may be key in the detatchment of methyl radical upon CID as Proton
transfers have been previously noted as the only method for certain reactions to
occur (Freeman et al. 1978). It has been postulated that the formation of
tetrahedral carbons within the ion trap drastically change the energy in which
substituents undergo any dissociation event. In this case, it is likely that an
intermolecular proton transfer occurs from C2 to N5, C2 to N8, and C6 to N1 in
1, 3, and 7-methyladeinine respectively, in order to weaken the CH3-purine
bond.

 

Figure 5. Exhibited above are the energetics involved in the loss of methyl
radical from each methyladenine derivative. Appearing from left to right are 7-
3- and 1-methyladenine with dissociation energies of 87.67, 91.22, 92.79 kcal
mol-1, respectively.

                                                                                                                     1-Methyladenine

     1-Methyladenine displays
the unique decay of the parent ion peak (m/z 148) into peaks at m/z 133 and m/z
107 upon NCE values increasing from 25-45 percent (Figure 6). The large peak
appearing at m/z 107 indicates a mass of 41 AMUs and is likely the result of a
severance event including CH3CN, and resulting in the detected
molecule C3H3N3-. Although each
spectrum contains a peak at m/z 133, a large aberration in the intensity is
clear in the spectrum for 1-methyladenine juxtaposed to that of 3- and 7- methyladenine.
Elucidation of the mechanism behind the unusual peak intensity is achieved by
computationally modeling the lowest energy path for the loss of CH3CN
compared to that of the methyl radical (Figures 5 & 7.) In spite of a .4
isolation width, a shoulder is detected at m/z 108 and m/z 149 due to the
possible formation of a radical 1-methyladenine species during the ESI process.

 

Figure 6. The fragmentation pattern of
1-methyladenine with respect to increasing normalized collision energy (NCE %)
is defined. Loss of the CH3CN fragment is emphasized. A peak
corresponding to the loss of methyl radical is seen developing in the center. A
shoulder is detected at 108 and 149 m/z due to the possible formation of a
radical 1-methyladenine species.

     A potential energy surface
describing the energetics involved in the complete dissociation of the acetonitrile
group from 1-methyladenine is obtained computationally (Figure 7). The
fragmentation event is first facilitated by the migration of the methyl
substituent to the C2 position forming a tetrahedral carbon and destabilizing
the bond between C2 and N5. This transition state of approximately 65 kcal·mol-1
yields a relatively low energy geometry optimization of 35 kcal·mol-1
relative to the methyladenine parent. The now weakened C-N bond then undergoes
a ring opening transition state occurring at 75.51 kcal·mol-1. The
resultant open ring compound rests at an energy of 55.59 kcal·mol-1.
At this juncture in the computing process several routes for the transfer of
H13 were explored. Transition states of reasonable energy were found for the transfer
of H13 to carbons 1, 3, and 5. In addition, proton transfers were conducted to
nitrogens 5 and 9. The optimized geometric structures of all the newly
protonated carbon species yielded unreasonable energies when juxtaposed to the
ion trap. All computed energies must fall within a range
that is accessible in the trap. When conducting a transfer to the N5 position a
high energy compound formed. Due to the relationship between HCN and the formation
of adenine partially through addition to 4-amino-5-cyanoimidazole (4-aminoimidazole-5-carbonitrile)17,
an attempt was made to lower the aforementioned energy by shuttling proton 12
to N5 in order to create a potentially stable NH2 moiety. This
approach lent a reasonable transition state energy of 82 kcal·mol-1
however, the newly formed NH2 containing species optimized to an
energy of 123.15 kcal·mol-1. In order to render these tribulations,
the excited state intermolecular proton transfer from C2 to N9 was analyzed.
Despite a long range atomic scan, the transition state was shown to occur at 95.56
kcal·mol-1 with an optimized geometric structure at 93.63 kcal·mol-1.
Although this is a high energy transition state and geometry, it has not been
unusual in the explorations of CID fragmentation involving adenine.14
The PES effectively describes the loss of CH3CN however, it is
hypothesized that a lower energy pathway exists explaining the relative intensities
of the m/z 107 and m/z 133 peak observed in the experimental result (Figure 6).  The lower energy pathway may include the
formation of the CH3NC isomer. Bond scans were employed again in
order to study the possibility of this occurrence.  The result of the bond scan between C3 and N5
yielded no discernable transition state, however, it is highly probably that
the separation occurs and possesses a low energy transition state.

3-Methyladenine

     The experimental CID evolution spectrum  with respect to increasing NCE value obtained
from 1-methyladenine provided curious results (Figure 8). Unlike the relatively
dynamic fragmentation patterns seen in 1- and 7- methyladenine this isomer displayed
the sole characteristic loss of methyl radical. It is evident that this arises  due to the position of the methyl subtituent
in the N3 position. DFT computations for 1- and 7-methyladenine show that excited
state intermolecular proton transfer to form tetrahedral intermediates gives
rise to the loss of alternative fragments. With the electron donating properties
of the CH3 subtituent it is likely that the aromaticity and electron
density provides a stabilizing effect in which only the methyl moiety can dissociate
before complete degradation of the molecule. Much like the experimental spectrum
for 1-methyladenine a small shoulder can be seen at the m/z 133 peak corresponding
to an NCE % of 30. This shoulder is resultant of the formation of a radical
3-methyladenine species during the ESI process.

Figure 8. The
spectrum above displays the fragmentation pattern of 3-methyladenine
with respect to increasing normalized collision energy (NCE%). Loss of methyl
radical fragment is emphasized.

Figure 7: The
potential energy surface for the loss of CH3CN from
1-methyladenine.

7-Methyladenine

     7-Methyladenine displays an experimental
spectrum (Figure 9) disimilar from both 1- and 3-methyladenine. A low relative
intensity peak is observed at m/z 121 representing a loss of
27 AMUs. This loss has been been observed in several anions previously studied
including adenine, guanine, oxazole, as well a many others13,14  and in nearly every case this mass has
correponded to an HCN molecule or one of it isomers.  The mechanism by which this severance occurs is
likely very similar to that of CH3CN from 1-methyladenine. It is
highly likely that a ring opening event occurs beteen N7 and C3 thus exposing
the HCN fragment as H5 N2 C7. In order for the ring opening event to take place
an intermolecular proton transfer of  H11
is probable. The energy of this dissociation is thought to be relatively high
due to the vast difference in intensity compared to the loss of methyl radical.
An alternative route to describe the fragment is the formation of the HNC isomer.
This event could likely involve an intermolecular proton tranfer from C2 or N9
to N7 in order to destabilize the N7-C3 bond. The destabilization of the bond will
likely result in a ring opening followed by dissociation of HNC as H11N9C3 or
H5N7C2. It Is once again significant to describe the formation of the
7-methyladenine radical during the ESI process observed as a shoulder in the
experimental spectrum. Attempts were once made to solve this problem by
reducing the isolation width of the spectrum to .4. Despite the small isolation
width the shoulder persisted to appear on the experimental spectrum. 

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