Eur. Phys. J. D (2012) 66: 13 DOI: 10.1140/epjd/e2011-20616-y
THE EUROPEAN PHYSICAL JOURNAL D
Colloquium
Metastable decay of DNA components and their compositions – a perspective on the role of reactive electron scattering in radiation damage ´ H.D. Flosad´ ottir, B. Omarsson, I. Bald, and O. Ing´ olfssona University of Iceland, Science Institute, Dunhagi 3, 107 Reykjav´ık, Iceland Received 25 October 2011 / Received in final form 17 November 2011 c EDP Sciences, Societ` Published online 25 January 2012 – a Italiana di Fisica, Springer-Verlag 2012 Abstract. Here we review recent studies on the metastable fragmentation of individual DNA and RNA building blocks and their compositions using matrix assisted laser desorption/ionization mass spectrometry (MALDI). To compare the fragmentation channels of small DNA components with larger compositions we have studied the metastable fragmentation of the deprotonated nucleobases, ribose, ribose-monophoshates, the nucleosides, the nucleoside 5 -monophosphates and selected oligonucleotides. Both previously published and unpublished data are reported. To gain a comprehensive picture of the fragmentation of individual components, metastable fragmentation of native components are in many cases compared to chemically modified components and isotopic labelling is used to unambiguously identify fragments. Furthermore, to shed light on the underlying fragmentation mechanisms we complement the experimental studies with classical dynamics simulations of the fragmentation of selected compounds. For the DNA and RNA components where dissociative electron attachment studies have been conducted we compare these to the metastable fragmentation channels observed here.
1 Introduction High-energy radiation is known to induce fragmentation of genomic DNA which can lead to cell apoptosis or mutation if not repaired correctly. Mutations can then further lead to cancer formation. For accurate radiation precaution protocols, as well as high quality radiation therapy methods, the processes causing mutations must be well understood on the molecular level. Until recently the focus of radiation damage studies was mainly on the role of radical species and reactive oxygen species (ROS) [1–3]. However, in the year 2000 low energy electrons (LEEs) were shown to resonantly cause single and double strand breaks in condensed DNA [4]. As LEEs are abundant along the radiation track (≈5×104 MeV−1 ) [5] this has triggered great interest in their role in the actual damage caused by high-energy radiation. In order to elucidate the underlying mechanism behind DNA and RNA damage caused by LEEs, dissociative electron attachment (DEA) measurements of the various, isolated nucleic acid components in the gas phase, have been performed. These include 2 -deoxyribose [6], ribose [7], the nucleobases [8–14], phosphoric acid esters [15], the nucleoside thymidine [16,17], ribose phosphate [17] and recently the nucleotide 2 -deoxy cytidine 5 -monophosphate [18]. Furthermore, considerable work has been committed to DEA of individual amino a
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acids and few studies have been conducted with dipeptides [19–24]. These DEA measurements on the DNA components have revealed how low energy electrons, even below 3 eV incident energy, can induce dissociation corresponding to DNA and RNA strand breaks, as well as multiple bond ruptures and rearrangement of atoms. The research focus has generally been on the primary dissociation mechanisms commonly observed from DEA. However, by moving from small molecules to more complex, biologically relevant molecules, the dissociation processes observed may deviate from the dissociations observed for the individual components, and rearrangement and metastable processes may play an increasing role [25–27]. Furthermore, due to the thermal instability of larger biological molecules, such as oligonucleotides and peptides, and the fact that thermal evaporation is most commonly used for gas phase DEA studies, the current gas phase DEA measurements are limited to the smaller components. Thus the question remains open if, and then how; the dissociation processes observed for the smaller components prevail in the actual biologically relevant macromolecules. The most pronounced dissociation channel, observed upon electron attachment to the nucleobases, the phosphoric acid esters as well as the nucleoside thymidine, is hydrogen abstraction forming the [M − H]− anion [9,12–14,16,28,29]. Similarly, the predominant dissociation mechanism of amino acids upon DEA of LEEs is also
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hydrogen abstraction [20,21,23,24]. Hydrogen abstraction has furthermore been shown to be an intermediate step before further, metastable dissociation of the nucleobase thymine at incident electron energies below 15 eV [30]. The further dissociation of the vibrationally activated [M − H]− anions formed through DEA may therefore be relevant in the damage induced by LEEs on nucleic acids. Here we present a short colloquium on recent results on the secondary fragmentation processes of [M − H]− ions of DNA components. Some of the work presented here has been published [31–34], but also additional new experimental data is presented. For the amino acid L-Valine, we have previously shown that the further fragmentation of the ion [M − H]− formed through DEA and by deprotonation depends on the site of hydrogen removal, but is independent of the formation mechanism of the [M − H]− ion [35]. Here we have utilized matrix assisted laser desorption/ionization (MALDI) to generate the deprotonated molecules in the gas phase, and to study the further, metastable decay of the deprotonated anions. Using MALDI, intact [M − H]− ions of large molecules can be generated in the gas phase. We have taken advantage of this, and studied the gas phase fragmentation of larger molecules than has been possible in DEA gas phase experiments. To shed light on the underlying mechanism of metastable nucleic acid fragmentation, we have studied the decay of the deprotonated nucleobases, sugars [31,32], nucleosides [33], sugar phosphates [34] and nucleotide monophosphates [34], constituting DNA and RNA, as well as the metastable decay of their composition to short, single stranded oligonucleotides [36]. For unambiguous correlation between the site of deprotonation and further fragmentation we have systematically blocked deprotonation sites by chemical modification in the nucleobase thymine [34] and the nucleosides uridine and guanosine [33]. Furthermore, for identification of bond dissociation selectivity and charge retention of the metastable decay of deprotonated sugars, 13 C- and D-isotope labelling was used. For thymine, the nucleosides and d-fructose we have also conducted classical dynamics simulations of the fragmentation of their deprotonated ions in order to gain insight into the dynamics and the fragmentation mechanisms responsible for the dissociations observed [33]. By this systematic approach, we have worked our way from the individual components towards the oligonucleotides, and compared the dissociation of the larger combined molecules with the free components. In this way we address the questions whether, and then how, fragmentation channels observed for the smallest compounds are passed on to the larger, combined nucleic acids. When appropriate, we compare the metastable decay observed for the deprotonated molecules with the previous DEA experiments on the same molecules. Furthermore, we use the experimental and theoretical results on the metastable dissociation of the individual components to deduce fragmentation mechanisms for the larger, combined polymeric molecules.
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Fig. 1. (Color online) Schematic representation of the MALDI instrument.
2 Methods 2.1 Metastable decay measurements Metastable decay of the deprotonated molecules was measured in negative ion, post source decay (PSD) mode with a commercial UV MALDI reflectron time of flight (ToF) mass spectrometer (Reflex IV, Bruker Daltonics, Bremen, Germany). Figure 1 shows a schematic representation of the instrument. In MALDI the sample molecules are generally co-crystallized, on a suitable substrate, with an excess amount of the matrix molecules. Typically the matrix molecules are small aromatic systems with acidic and/or basic functional groups. The substrate with the sample is introduced into the ToF mass spectrometer where it serves as the repeller in a typical Wiley-McLaren double focusing ToF setup. The sample is then irradiated with nanosecond (ns) pulses of a focused UV laser causing explosive evaporation of matrix and sample molecules. In the expanding plume of the evaporated molecules both deprotonated and protonated, analyte and matrix molecules are formed (the ionization in MALDI proceeds mainly through proton transfer). After a delay of a few hundred ns with respect to the desorption/ionization laser, the ions are extracted with a high voltage pulse into a field free flight tube and the flight time is measured. The pulsed delayed extraction is used to compensate for the ion energy spread to achieve better ion focusing. To study the fragmentation during the flight through the field free region, i.e. the metatable decay, the instrument is run in post source decay (PSD) mode. In the PSD mode a single m/z ratio is selected from the ensemble of ions by an ion gate placed about 75 cm down stream of the linear flight tube. The ion gate is a simple deflector which is normally closed (deflecting all ions) but is pulsed to ground potential to allow ions to pass that have arrival times corresponding to the m/z ratio of the ions of interest. The fragmentation during the field free linear flight (125 cm) is then analysed with a gridless reflectron at the end of the linear flight tube. The field free flight tube after the reflectron is about 75 cm long. In the current study the ions were generated with an N2 -laser at 337 nm. The repetition rate was 7–10 Hz and the laser power was kept about 20% above the detection
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threshold for the corresponding parent ions. To average out sample inhomogeneity the laser spot was moved manually over the sample during acquisition. The negative ions were extracted in pulsed delayed extraction mode with a delay time of 200 ns and accelerated into the reflectron ToF mass spectrometer. In the PSD studies, the mass gate was set at ±5Da and the total acceleration voltage into the field free linear region was 25 kV. This results in about 6–26 μs flight time depending on the ion mass to charge ratio. This is the time window within which we observed metastable decay. To assure for observation of all fragments, the reflectron voltage was stepped down in 7–9 segments, depending on the sample. Individual segments are the sum of 500 shots, which were recorded by using the fragmentation analyses and structural ToF method FAST, within the instrumental control software r . The alignment of individual segments and FlexControl the mass calibration of the spectra was carried out with r software also provided by the instruthe FlexAnalysis ment manufacturer. 2.2 Sample preparation The compounds studied, cytosine, uracil, thymine, 1N methyl thymine (1), guanine, adenine, d-ribose, d-fructose, uridine, guanosine, adenosine, cytidine, thymidine, 2 -deoxy guanosine, 2 -deoxy adenosine, 2 -deoxy cytidine, ribose 5-monophosphate sodium, 2-deoxyribose 5 -monophosphate sodium, 2 -deoxy thymidine monophosphate (dTMP), 2 -deoxy guanosine monophosphate (dGMP), 2 -deoxy adenosine monophosphate (dAMP) and 2 -deoxy cytidine monophosphate (dCMP) were purchased from Sigma-Aldrich, Germany as high purity samples (>98%) and used without further purification. The thymine derivative 3-N methyl thymine (2), uridine derivatives; N-methyl uridine (3), 2 ,3 -O-isopropylidene uridine (4), 5 -O-methyl 2 ,3 -O-isopropylidene uridine (5) and 2 ,3 -O-isopropylidene N-methyl uridine (6) and the modified guanosines; N-methyl guanosine (7), 2 ,3 -Oisopropylidene guanosine (8), and 2 , 3 -O-isopropylidene 5 -O-methyl guanosine (9) were prepared in-house from the respective nucleosides and bases. The preparation and purification of 2 was performed as described by Breeger et al. [37] and H-NMR was used to verify the molecular structure. The preparation and purification protocols for 3–9 have been described elsewhere [33]. The hexameric oligonucleotides were synthesized and purified in-house as described by Stano et al. [36]. After purification the samples were dried on a Thermosavant ISSI110 speedvac system and redissolved in 100 μL sterile purified water. The concentration was determined on a 500 times diluted sample by UV absorption at 260 nm. The samples were then separated into 10 μL portions and stored at −20 ◦ C until use. For the MALDI PSD measurements, samples of the DNA building blocks were prepared by spotting 0.5 μL of a 1 mg/mL aqueous solution of the matrix bisBenzimide H 33258; C25 H24 N6 O·3HCl·xH2 O (98%, Sigma-Aldrich, Germany) on a stainless steel sample carrier and let dry
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under atmospheric pressure and at room temperature (RT). The sample solution was then spotted (0.5 μL of a 10 mg/mL MeOH) on top of the matrix crystals, and let dry at atmospheric pressure and at RT. BisBenzimide is not a conventional MALDI matrix but has proven very useful for low mass PSD experiments as it does not fragment to form m/z ratios that interfere with the ion selection in the low mass range [31]. The aqueous hexameric oligonucleotides (2 μL, 0.1 mmol/mL) were mixed with the matrix DHB (2 μL, 10 mg/mL) and acetonitrile (2 μL) and spotted in droplet size of 0.5 μL on a r sample plate and let dry under 400 μm AnchorChip atmospheric pressure at RT. 2.3 Classical dynamics simulations The calculations were carried out using density functional theory (DFT) with a plane wave basis set (energy cutoff at 395.994 eV) and the PW91 functional as implemented in the VASP code [38,39]. For the plane wave basis set, periodic boundary conditions need to be enforced. In these simulations the box was chosen to be 18 ˚ A to ensure for negligible interactions between periodic images. As the plane wave basis set describes all parts of the simulation box equally well and atomic movements do not cause changes in the basis functions, such a basis set is convenient for molecular dynamics. To ensure for zero total charge within the system, which is necessary in this periodic approach, a uniform background charge was introduced to compensate the charge in the simulations of the deprotonated anions. The background charge does not affect the forces acting on the atoms. The simulation steps have been described in detail elsewhere [33] but some of the important features are noted here for completeness. First, the molecular geometry of the neutral molecules was optimized using damped molecular dynamics, and when the force had become smaller than 0.3 ˚ A/eV the conjugate gradient algorithm was used. To assure for the stability of the deprotonated molecules the same procedure was applied. After geometrical optimisation the neutral geometry was given internal energy corresponding to 298 K by scaling the atomic velocities, and the vibrational modes were allowed to equilibrate over 1000 fs with microcanonical classical dynamics using the Verlet algorithm to integrate Newtons equations of motion. The simulations were allowed to continue for a further 1000 fs in order to create a set of 10 configurations of 100 fs interval. The individual configurations resulting from this procedure were used as starting geometries for further simulations (10 for each molecule and deprotonation site). Before such further simulations were run, an internal energy of 8 eV was added to each configuration by scaling the atomic velocities. This was done to account for the excitation energy acquired in the MALDI process. Then a hydrogen was removed from the respective deprotonation site and an additional charge was defined within the system. Finally, a constant energy (microcanonical) simulation of the vibrationally excited, deprotonated anions in their ground electronic state was carried out for 500 fs using the Verlet algorithm to again
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integrate Newtons equations of motion. For each deprotonation site, all ten initial configurations were used as starting geometries resulting in ten classical dynamics simulations for each molecule and each deprotonation site. The dissociations, which occurred during this simulation period were documented and the charge of each fragment was determined by Bader’s method [40–42]. These simulations are only a first order representation of the experimental conditions and the internal energy added (8 eV), is higher than what has been estimated for small molecules in MALDI, 4–5 eV [43,44]. In the current measurements, the observation time after mass selection is about 8–12 μs for the smaller molecules and about 20 μs for the oligonucleotides. The computational effort for such long trajectories is excessive. The higher internal energy is therefore chosen to accelerate the fragmentation so that it can be observed in shorter trajectories. It is, however, important to be aware that increasing the internal energy may also open new fragmentation channels and change the ratio between different channels. To elucidate the fragmentation mechanisms responsible for the metastable dissociation of the deprotonated molecules, and to test the ability of the simulation approach to predict fragmentation products, we simulated the dynamics of thymine, 1N methyl thymine, 3N methyl thymine, uridine and 2 -deoxy guanosine after deprotonation at the various sites.
3 Results and discussion The nucleic acids DNA and RNA are composed of polymeric chains of nucleosides consisting of the furanose form of the pentose 2 deoxyribose and ribose, respectively, and connected by phosphodiester bonds bridging the 3 and the 5 end of the sugars. The nucleobases cytosine, thymine, uracil, adenine and guanine, which constitute the variable units in the nucleic acids, are connected to the sugar moiety by a β-glycosidic bond to the 1 -C. Figure 2a shows the structural composition of a nucleoside 5 -monophosphate as either the ribonucleotide (R = OH) or the 2 -deoxyribonucleotide (R = H). For clarity the individual components discussed in the proceeding text are signified with over-braces. The naturally occurring DNA and RNA nucleobases are depicted in Figure 2b. The 2 -deoxynucleotides cytidine, thymidine, adenosine and guanosine are the principal components of DNA and similarly the ribonucleotides cytidine, uridine, adenosine and guanosine are the principal components of RNA. In the following discussion [M − H]− refers to the respective deprotonated molecular ion of the nucleosides and nucleotides. For the deprotonated bases and sugars (ribose), [B − H]− and [R − H]− is used, respectively, and B and R signifies the corresponding neutral species. In the context of further fragmentation, however, the deprotonated molecule is denoted as (M − H) for the larger ions as well as the bases and sugars. In this system a deprotonated molecule (nucleoside or nucleotide) that has lost the neutral base is signified as [(M − H) − B]− . A depro-
Fig. 2. The molecular structure of (a) a generic ribonucleotide 5 -monophosphate (R = OH) and 2 -deoxynucleotide 5 -monophosphate (R = H), and (b) the naturally occurring nucleobases that constitute the DNA and RNA building blocks.
tonated molecule (base, nucleoside or nucleotide) that has lost a HNCO unit is signified as [(M − H) − HNCO]− . The number of possible sequences for a moderately long DNA or RNA chain are immense, and study of processes within the nucleic acids at the molecular level can be very complicated. To reduce the complexity of the problem it is thus often necessary to take a “bottom-up” approach and study the individual components separately and use the information attainable to draw conclusions about the behaviour of their composites.
3.1 Metastable fragmentation of deprotonated nucleobases The nucleobases can be categorized into two groups by their chemical structure; purine and pyrimidine bases, and we will discuss these groups separately. Figure 3 shows the PSD spectra of the deprotonated pyrimidine nucleobases cytosine, uracil and thymine. A prominent peak at 42 amu is observed from all pyrimidine bases, which
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a) Cytosine NCO
[(M-H)-HNCO]
-
-
*
x10
[M-H]-
b) 3N-methylthymine (2)
b) Uracil
*
c) Thymine
40
[M-H]-
a) 1N-methylthymine (1) NCO[(M-H)-HNCO]-
40
60
80 m/z
100
120
Fig. 3. Post source decay spectra of the pyrimidine nucleobases; cytosine (a), uracil (b) and thymine (c). Same fragments from different molecules are labelled and connected with a dotted line. The deprotonated pyrimidine nucleobases fragment to form NCO− and [(M − H) − HNCO]− .
60
80
100 m/z
x10
120
140
Fig. 5. Metastable decay spectra of the deprotonated methylated thymine derivatives 1 (a) and 2 (b). Deprotonation from 3N–H (a) results in the formation of NCO− and [(M − H) − HNCO]− . Deprotonation from 1N–H does not induce further fragmentation. An asterisk is used to label background peaks.
Fig. 6. Proposed dissociation mechanism for the formation of NCO− from the deprotonated pyrimidine nucleobases (shown for thymine). The NCO− formation after 3N–H deprotonation is proposed to proceed through reverse pericyclic cycloaddition.
Fig. 4. The two thymine derivatives 1N methyl thymine (1) and 3N methyl thymine (2). For 1, deprotonation can only occur at the 3N–H site and for 2 deprotonation only at the 1N–H site.
is assigned to the fragment ion NCO− . This fragment ion has been observed earlier from DEA to the pyrimidine bases [11,28] and in metastable decay of uracil in combination with Cu2+ ions, where it was shown by isotope labelling to include the 3N and the 2C=O atoms [45]. The second channel observed for cytosine and thymine is the formation of the fragment [(M − H) − HNCO]− which is complementary to NCO− with respect to the retention of the proton. Pyrimidine nucleobases have two acidic protons, one at 1N and one at 3N of the ring. These are the two main sites of deprotonation, which in turn lead to the formation of two distinctly different anions. In DEA, hydrogen abstraction from the pyrimidine nucleobases has been shown to proceed through distinct resonances, characteristic for these individual sites [12,13]. To elucidate the role of the individual deprotonation sites in metastable decay of the deprotonated bases, we have measured the fragmentation of the methylated thymine derivatives; 1N methyl thymine (1) and 3N methyl thymine (2) (see Fig. 4). The former
can only deprotonate at the 3N–H site and the latter only at the 1N–H site. Their PSD spectra are shown in Figure 5. Like the native thymine, 1N-methyl thymine dissociates to form the fragments NCO− and [(M − H) − HNCO]− , while deprotonated 3N-methyl thymine does not show any further fragmentation. We thus propose a fragmentation mechanism for the NCO− formation from the pyrimidine bases, which is initiated by 3N–H deprotonation (see Fig. 6). This fragmentation mechanism is similar to a reverse pericyclic cycloaddition, but in this case the additional charge stabilizes the fragmentation products. This mechanism also explains the selectivity of 3N–2CO− since the 1N−2C and 3N−4C are the only bonds that can be ruptured via a reverse pericyclic reaction. To further support the proposed mechanism we performed classical dynamics simulations on the native thymine and the derivatives 1N methyl thymine (1) and 3N methyl thymine (2) deprotonated at the 3N–H and 1N–H site, respectively. Figure 7 shows an example of the NCO− formation observed in the simulations for (a) the native thymine and (b) 1N methyl thymine (1) when these are deprotonated at 3N. The simulations on native thymine deprotonated at 3N, showed NCO− formation in four out of the ten (4/10) simulations via reverse pericyclic cycloaddition but only one out of ten (1/10) simulations of native thymine deprotonated at 1N, showed NCO− formation. For the thymine derivatives nine out of the ten (9/10) simulations of deprotonated 1N methyl thymine resulted in NCO− formation,
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Fig. 7. (Color online) To the left, snapshots from the classical dynamics simulations for the nucleobase thymine deprotonated at the 3N site (a) and the modified nucleobase 1N methyl thymine deprotonated at the 3N site (b). Carbon atoms are gray, oxygen red, nitrogen blue and hydrogen white. To the right of this figure a schematic view of the dissociation mechanism is given. a) Adenine
[(B-H)-CHN]
-
[B-H]
-
-
[(B-H)-CH2N2]
[C2N2H]
x10
b) Guanine
[(B-H)-CH3N2/HNCO]
[C2HN3O]
-
[(B-H)-CH2N2/NCO]
-
[(B-H)-NH3/OH]
-
[B-H]
-
x10
40
60
80
100 m/z
120
140
Fig. 8. Metastable decay spectra of the deprotonated purine nucleobases adenine (a) and guanine (b). Only low yield fragment mass peaks are observed, indicating the higher stability of the deprotonated purine bases compared to the pyrimidine bases.
but no trajectory from deprotonated 3N methyl thymine resulted in fragmentation. The PSD spectra of the purine nucleobases adenine and guanine are shown in Figure 8. These are characterized by several low yield mass peaks, whose m/z ratios correspond to the elimination of HCN, H2 CN2 and HC2 N2 from adenine and OH (NH3 ), NCO (H2 CN2 ), HNCO (H3 CN2 ) and H2 C3 NO from guanine. In addition we also observe the m/z ratio 26 from adenine, which we attribute to CN− rather than C2 H− 2 formation. Gas phase guanine has been shown to exist as the enol-tautomer rather than the keto-tautomer, which is the form when guanine is incorporated into DNA [46]. Hence, the heterocyclic structure of the deprotonated guanine in the gas phase is expected to be aromatic. The same is true for deprotonated adenine, which might explain the observed stability of the deprotonated purine bases. In electron attachment to the nucleobases, no stable parent ion is observed. The most pronounced DEA frag-
ment for all the nucleobases except guanine is the molecular ion after loss of one hydrogen atom [M − H]− . This fragment is observed at electron energies below 3 eV [9,11], and has been shown to be formed in thymine and uracil with high site selectivity depending on the resonances involved. At incident electron energy close to 1.0 eV the hydrogen loss proceeds from 1N, whereas a resonance located at 1.8 eV results in hydrogen loss from 3N. In the case of DEA to adenine, hydrogen loss occurs exclusively from 9N at electron energies below 1.5 eV [47]. At higher incident electron energies both the pyrimidine and the purine bases have been shown to dissociate by cross ring cleavage resulting in a variety of fragment ions [9,48–50]. However, in a study of DEA to guanine a very low yield of [G − H]− is reported indicating that guanine decomposes in the thermal evaporation process commonly used in DEA experiments [8,9]. The assignment of individual fragments from guanine as DEA products from the intact base is therefore not clear. The NCO− formation from the pyrimidines is consequently the only fragmentation channel that we can unambiguously identify as being in common with the fragmentation of the deprotonated bases in MALDI PSD and in DEA. It should, however, be mentioned that the CN− formation is a prominent channel in DEA to adenine and HCNO loss is also observed from the guanine molecular anion. In summary we can conclude that the dominating fragmentation channel of the pyrimidine bases is the siteselective formation of NCO− as well as the elimination of HNCO from 3N–H deprotonation. The deprotonated purine nucleobases, are generally very stable due to their aromatic character, and are thus unlikely to directly contribute significantly to DNA fragmentation. 3.2 Metastable fragmentation of deprotonated D-ribose Central building blocks of the DNA and RNA backbone are the sugars 2-deoxy-d-ribose and d-ribose, respectively,
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a)
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b) H
HO H
HO
H
C5
HO
H OH
OH
C1 OH H
β-D-Ribopyranose
c)
C5 H H HO
O
OH H C1 H OH
β-D-Ribofunaronse
0,2X
4
5
3
2
O OH 0 1
HO OH 0,2
OH
A
Fig. 9. The molecular structure of β-d-ribose in its pyranose form (a) and β-d-ribose in its furanose form (b). The carbon atoms 1C and 5C are signified in both the pyranose and furanose forms. (c) Shows an example of the nomenclature introduced by Domon and Costello for fragmentation of deprotonated monosaccharides [52]. In X-type cleavage the charge retention is on the fragment containing the anomeric center (1C) and in A-type cleavage the charge retention is on the fragment containing the 5th carbon. The numbers in superscript denote the bonds dissociated as shown in the figure.
and a fragmentation through cross ring cleavage of the sugar unit can correspond to a direct DNA strand break. Bound within the backbone the sugars adopt the furanose form, while the isolated sugars crystallize in the pyranose form, which is most likely preserved during laser desorption [51]. Figure 9 shows the molecular structure of d-ribose in the pyranose form (a) and the furanose form (b), along with an example of the notation of cross-ring cleavage according to Domon and Costello [52]. In this nomenclature X-type fragment ions contain the anomeric centre, while A-type fragment ions contain the 5th carbon atom. Any superscript numbers then denote the bonds dissociated as shown in Figure 9. Under the current experimental conditions the deprotonated 2 deoxyribose was formed with too low intensity to reliably measure its metastable fragmentation. Thus, metastable decay has only been measured for deprotonated d-ribose [31]. Like other sugars, d-ribose contains a sequence of CHOH units and unambiguous identification of individual fragments by mass spectrometry alone can thus be problematic. To address this problem we studied the metastable decay of the isotope labelled riboses 1-13 C-d-ribose, 5-13 C-d-ribose and C-1-D-d-ribose in addition to the native ribose [31]. Figure 10 compares the PSD spectra of the native ribose (a), with that of 5-13 C-d-ribose (b), 1-13 C-d-ribose (c) and C-1-D-d-ribose (d). In the metastable decay of deprotonated d-ribose; [R − H]− , we observe the typical sugar fragmentation, which is characterized by the loss of water and formaldehyde units. The main fragmentation
Fig. 10. Metastable decay spectra of deprotonated d-ribose (a), 5-13 C-d-ribose (b), 1-13 C-d-ribose (c) and C-1-D-d-ribose (d). When the peaks are shifted by one mass to higher masses, the anionic fragment contains the isotope labelled atom. Peaks labelled with an asterisk are background peaks. See text for the discussion.
channels observed for the native [R − H]− are the elimination of a single H2 O (m/z = 131), loss of a single CH2 O unit (m/z = 119), loss of CH2 O, H2 O and H (m/z = 100), loss of two CH2 O units (or C2 H4 O2 ) (m/z = 89), and the loss of two CH2 O units and one H2 O (m/z = 71) [7,31]. From these channels the C3 H5 O− 3 formation (m/z = 89) is by far the most dominant product anion. In the PSD spectra from 1-13 C-d-ribose (c), and C-1D-d-ribose (d) the fragments appearing at m/z = 119 and m/z = 100 in native d-ribose appear shifted by one mass unit. In the PSD spectra from the deprotonated 5-13 C-dribose, on the other hand, these fragments appear exclusively at the same m/z ratio as from the native d-ribose. Hence, the loss of one CH2 O unit (m/z = 119) and the loss of CH2 O, H2 O and H (m/z = 100) proceeds exclusively from the 5 end, i.e., these fragments are both formed through a 0,4 X cross ring cleavage. The fragment associated with the loss of C2 H4 O2 and one H2 O (m/z = 71) (or two CH2 O and one H2 O), on the other hand, is predominantly shifted to m/z = 72 in the PSD spectra of 5-13 Cd-ribose but appears mainly at m/z = 71 in the spectra of the other isotopic labelled analogues. Hence, this fragment is predominantly formed through carbon loss from the 1-C end and constitutes a 0,2 A cross ring cleavage. Judging from the high site selectivity, most likely a C2 H4 O2 unit is eliminated in this reaction.
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Fig. 11. (Color online) Proposed fragmentation mechanism for the formation of the m/z ratios 119, 89 and 71 from deprotonated d-ribose. Two channels are proposed to be open; one constitutes neutral loss from the 5-C end and leads to the m/z ratios 119 and 89, the second constitutes neutral loss from the 1-C end and leads to the m/z ratios 89 and 71. The neutral loss from the 1-C end leads to a X-type fragment with m/z = 89 and neutral loss from the 5-C end leads to a A-type fragment with the same m/z-ratio. Reprinted from [31] with permission from ScienceDirect.
The most pronounced fragment in the PSD spectra of the native ribose appears at m/z = 89 and is assigned to 13 the sum formula C3 H5 O− 3 . In 5- C-d-ribose about 2/3 of the signal intensity is shifted from m/z = 89 to m/z = 90 and in 1-13 C-d-ribose and C-1-D-d-ribose about 1/3 of the signal intensity is shifted to m/z = 90. The most intense signal in the PSD spectra from the native d-ribose, is thus composed of at least two different, but isobaric fragments. We attribute these to a 0,2 A cross ring cleavage and a 0,3 X cross ring cleavage, which proceed from elimination of the 1-C and 5-C ends respectively (see Fig. 9). We cannot exclude a contribution from H2 CO loss from both sides, but this must be considered an unlikely process, as it would occur through two separate events. Figure 11 shows a reaction scheme for the ring opening of d-ribose deprotonated at the anomeric hydroxyl group, leading to m/z = 119 through 0,4 X cross ring cleavage and the two possible re-
action sequences (X and A) yielding the m/z = 89 and 71 anions. The use of isotope labelled d-ribose analogues showed that two different fragmentation pathways are operative, with charge retention either on the fragment containing the anomeric centre (1-C) or at the opposite end (5-C). This is in contrast to previous CID studies where only A-type cross-ring cleavage was proposed [53]. The heavier fragment anions at m/z = 119 and m/z = 100 are exclusively formed through X-type cleavage and contain the anomeric centre (1-C), whereas the smaller fragment anion at m/z = 71 predominantly arises from an A-type cleavage with the negatively charged fragment still containing the 5-C terminus of the sugar. The most prominent fragment, m/z = 89, on the other hand, is formed through both A- and X-type cleavage. This site selectivity of individual fragmentation channels is most likely in common with
Eur. Phys. J. D (2012) 66: 13
all monosaccharides, since we observed similar tendencies in a detailed experimental and computational study on d-fructose [32]. Furthermore, for d-fructose we could also show that different deprotonation sites can result in different fragment anions with the same m/z ratios and that the sugar fragmentation is generally composed of two or all three of the following reaction steps; (i) intramolecular proton transfer, (ii) anomeric ring opening and (iii) antiperiplanar dissociation. The proton transfer barrier between the individual hydroxyl groups in the sugar is low and the anomeric hydroxyl group is the most acidic. The dominant starting point for metastable fragmentation is thus likely to be deprotonation at the anomeric centre leading to ring opening and further carbon abstractions. The fragmentation of d-ribose and d-fructose via DEA proceeds within two resonances, a narrow one near 0 eV and a broader resonance at energies between 6 and 9 eV [54]. For both sugars the contribution through the higher energy resonance is considerably less than through the 0 eV resonance. Similar to the metastable decay of the deprotonated sugars, fragmentation upon DEA is in general characterized by elimination of one or more neutral H2 O and H2 CO units (Cn H2n On ) [7,31]. Furthermore, in DEA to d-ribose the same neutral loss is observed for the m/z ratios 131 (H2 O loss), 119, 100 and 71 as is observed in the PSD spectra. Hence these appear predominantly at m/z = 132, 120, 101 and 72 in DEA to d-ribose. In DEA, as well as in PSD, these fragments must in most cases result from several bond ruptures and new bond formations, but it can generally not be determined if these proceed in one step or through a sequence of low energy barrier channels. Nonetheless, like in the metastable decay, distinct site selectivity with preferable carbon loss from the 5-C end is observed in the DEA experiments. The m/z = 89 species is the only fragment that predominantly appears at the same m/z ratio in DEA and PSD (i.e., not shifted by one mass unit). This fragment appears with low intensity in DEA where it was suggested to be formed through a sequential reaction [31]. This involves initial hydrogen abstraction through DEA to form [R − H]− and subsequent metastable decay of the [R − H]− anion. 3.3 Metastable fragmentation of deprotonated nucleosides and 2 -deoxynucleosides A nucleoside contains one base and one sugar unit connected via a β-glycosidic bond (Fig. 2), and can thus be seen as the first step in combining the individual DNA components. The metastable dissociation of the deprotonated ribonucleosides and 2 -deoxynucleosides are shown in Figures 12 and 13, respectively. There are two main fragmentation channels that are in common for all the deprotonated nucleosides (ribo and deoxy). These are the glycosidic bond rupture creating the anionic (deprotonated) base [B − H]− (and the anionic (deprotonated) ribose [R − H]− for some nucleosides), and the 0,2 X sugar cross ring cleavage creating the fragment [B + 42]− from the ribonucleosides and [B + 26]− from the 2 deoxynucleosides.
Page 9 of 20
a) Cytidine NCO-
0,2
[B-H]138
[(M-H)-HNCO]-
X
174 182
5x
b) Uridine NCO-
[(M-H)-B]10x
c) Adenosine
[B-H]-
10x
d) Guanosine
10x
40
60
80
100
120
140
160
180
200
m/z Fig. 12. Metastable decay spectra of the deprotonated nucleosides; cytidine (a), uridine (b), adenosine (c) and guanosine (d). The main fragmentation channels in common for all the nucleosides are the glycosidic bond rupture and the 0,2 X sugar cross ring cleavage. The pyrimidine containing nucleosides (a and b) additionally fragment to form NCO− and [(M − H) − HNCO]− . Same fragments from different molecules are labelled and connected with a dotted line. Reproduced from [33].
In the nucleosides the combination of a base with the sugar through the glycosidic bond removes the anomeric hydroxyl group of the sugar. Furthermore, the sugar in the nucleosides adopts a furanose structure in contrast to the pyranose structure expected for the isolated monosaccharides in the gas phase [51]. Nonetheless, in the nucleosides the only observed sugar fragmentation is the 0,2 X cross ring cleavage which is complementary to the 0,2 A cross ring cleavage with respect to the charge retention. This is the most pronounced bond rupture observed in metastable decay of deprotonated ribose in the gas phase and leads to m/z = 89 [31]. In the nucleosides, the 1N and 9N deprotonation sites are additionally removed from the pyrimidine and purine bases, respectively. However, also here the same main base fragmentation is observed for the pyrimidine containing nucleosides as was observed for the isolated pyrimidine bases, i.e. NCO− and [(M − H) − HNCO]− formation. This is in good agreement with the findings that these channels are initiated through deprotonation at 3N of the pyrimidine bases [33]. The nucleosides and 2 -deoxynucleosides have several potential deprotonation sites and, as has been shown
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Eur. Phys. J. D (2012) 66: 13
Fig. 13. Metastable decay mass spectra from deprotonated 2 -deoxynucleosides; 2 -deoxy cytidine (a), thymidine (b), 2 deoxy adenosine (c) and 2 -deoxy guanosine (d). The main fragmentation channels for all 2 -deoxy nucleosides are the glycosidic bond rupture and 0,2 X sugar cross ring cleavage. The pyrimidine containing nucleosides (a and b) additionally fragment to form NCO− and [(M − H) − HNCO]− . Same fragments from different molecules are labelled and connected with a dotted line. Reproduced from [33].
clearly for the isolated thymine base, the initial deprotonation site determines the proceeding fragmentation steps [34]. Furthermore, in DEA it has been shown that hydrogen abstraction from the pyrimidine bases is highly site selective with regards to individual resonances. In this context we synthesised the modified uridine derivatives (3–6) and the guanosine derivatives (7–9) shown in Figure 14. In these compounds we have selectively blocked individual deprotonation sites to be able to unambiguously identify which deprotonation sites lead to which fragmentation channels. Complementary molecular dynamics simulations of the different deprotonated uridine and 2 -deoxyguanosine derivatives were also used, to elucidate the respective fragmentation mechanism [33]. We will discuss the results for uridine as an example here, but similar results were also obtained for guanosine [33]. Figure 15 shows the PSD mass spectra of the native deprotonated uridine compared with the deprotonated uridine derivatives 3–5. Only sugar cross ring cleavage and glycosidic bond rupture are observed upon sugar deprotonation (panel (b)), while base deprotonation (panels (c) and (d)) results in NCO− formation and HNCO elimination
Fig. 14. Molecular structure of the synthesized uridine (3–6) and guanosine derivatives (7–9). These are: 3N metyl uridine (3), 2 ,3 -O-isopropylidene uridine (4), 2 ,3 -O-isopropylidene, 5 -O methyl uridine (5), 2 ,3 -O-isopropylidene, 3N methyl uridine (6), 3N methyl guanosine (7), 2 ,3 -O-isopropylidene guanosine (8) and 2 ,3 -O-isopropylidene, 5 -O methyl guanosine (9).
but no sugar fragmentation. The simulations for the statistical dissociation of uridine are in accordance with the experiments and predict three fragmentations; (i) a glycosidic bond rupture creating [B − H]− , (ii) a 0,2 A cross ring cleavage that is observed upon sugar deprotonation, and (iii) NCO− formation upon base deprotonation. Figure 16 shows the fragmentation mechanisms for the different deprotonation sites as revealed by the simulations of deprotonated uridine. In the simulations, the 0,2 X cross ring rupture (Fig. 16c) is observed after 3 -OH deprotonation followed by aldehyde formation, 2 -C-3 -C bond rupture, 1 -C-2 -C double bond formation and 1 -C–O cleavage. For the sugar cross ring cleavage the simulations predict the bond ruptures correctly, but the charge retention is not well described. In the experiment 0,2 X cross ring cleavage
Eur. Phys. J. D (2012) 66: 13
Fig. 15. Metastable decay mass spectra of the deprotonated native uridine (a) and its derivatives 3–5 (b–d). Deprotonation at the 2 -OH and 3 -OH (b) is found to induce glycosidic bond rupture and 0,2 X sugar cross ring cleavage. Deprotonation at 3N–H (c and d), on the other hand, leads to NCO− and [(M − H) − HNCO]− formation. Same fragments from different molecules are labelled and connected with a dotted line. Reproduced from [33].
is observed but the simulations show the charge retention on the complementary fragment. Hence, the simulations predict a 0,2 A cross ring cleavage as is observed for the isolated ribose. Similarly the simulations predict NCO− formation after deprotonation at 3N of the base (Fig. 16a). This is in good agreement with experiments, however, in the experiments elimination of neutral HNCO from the parent ion is also observed. In the simulations, the glycosidic bond rupture is observed via three fragmentation channels. One includes an aldehyde formation and 2 -C-3 C bond rupture. The other two pathways occur via epoxide formation from, either 2 -C–O− to 1 -C or from 3 -C– O− to 4 -C. The first leads directly to a glycosidic bond rupture, while the second leads to a 4 -C–O sugar ring rupture accompanied by aldehyde formation and a subsequent glycosidic bond rupture as shown in Figure 16b. In all cases the sugar cross ring cleavage and the glycosidic bond rupture result from deprotonation at the 2 -OH or 3 -OH sites, but these are identical since the 1,2-diol arrangement allows for low-energy barrier proton sharing between the two hydroxyl groups (0.59 kcal/mol [55]). In summary, the main fragmentation channels observed in the metastable decay of the isolated ribose and the bases in the gas phase are still operative in the nucleosides. In addition a new channel opens up, which con-
Page 11 of 20
Fig. 16. Proposed fragmentation mechanisms for deprotonated uridine as observed from the classical dynamics simulations; (a) 3N–H deprotonation induces base fragmentation as previously observed for thymine. (b) Glycosidic bond rupture is observed to occur through three different fragmentation channels, all initiated by 2 -OH or 3 -OH deprotonation. One of the three mechanisms is shown here. (c) 0,2 X sugar cross ring cleavage is observed upon 3 -OH deprotonation.
stitutes the rupture of the glycosidic bond that connects the sugar to the base. By blocking individual deprotonation sites it was shown unambiguously that the base fragmentation results exclusively from deprotonation at 3N of the base in the pyrimidine containing nucleosides, but no base fragmentation is observed from the purine containing nucleosides. Sugar cross ring cleavage and glycosidic bond rupture, on the other hand, results exclusively from deprotonation of the sugar moiety. The classical dynamic simulations conducted for the nucleosides were found to accurately reproduce the bond ruptures initiated from the different deprotonation sites, however, the charge retention did often not correspond to the experimental observations. In thermal evaporation the nucleosides have a strong tendency to decompose. Thus reliable DEA experiments with nucleosides in the gas phase are limited to a study on the nucleoside thymidine. Here, hydrogen abstraction was observed through resonances close to 2 and 4 eV, respectively [16], but no further fragmentation was observed through these resonances. Glycosidic bond rupture creating anionic thymine [T − H]− , on the other hand, is only observed through a core excited resonance close to 6 eV [17]. Other fragments were not observed in these experiments.
Page 12 of 20
Eur. Phys. J. D (2012) 66: 13
a) Ribose monophosphate PO4H2-
0,3
0,2
A 0,2
PO3-
A
[(M-H)-H2O]-
A-OH
b) 2’-deoxyribose monophosphate
[(M-H)-2H2O]-
60
80
100
120
140 m/z
160
180
200
Fig. 17. Metastable decay mass spectra from the deprotonated sugar phosphates ribose monophosphate (a) and 2 deoxyribose monophosphate (b). The fragments in common for both molecules are labelled and connected with dotted lines.
3.4 Metastable fragmentation of deprotonated sugar phosphates The combination of a phosphate group with either ribose or 2-deoxyribose, to form the respective sugar phosphates (Fig. 2), gives the smallest unit of the RNA and DNA backbone respectively. To study the metastable decay of the backbone without the influence of the bases, we have measured the metastable fragmentation of the deprotonated ribose 5-monophosphate (RP, m/z = 229) and 2deoxyribose 5-monophosphate (dRP, m/z = 213). Figure 17 shows the mass spectra of RP and dRP. Similar to the nucleosides, preservation of the most pronounced fragmentation channels in the isolated sugar in the gas phase is also observed in both the deoxy- and the ribose monophosphate. The typical water loss generally observed in both DEA and metastable decay of isolated sugars is a major channel in the metastable decay of both RP and dRP. In dRP the second most intense fragmentation observed, is the loss of one water unit to form [(M − H) − H2 O]− (m/z = 195). From RP, on the other hand, the loss of two water units to form [(M − H) − 2H2 O]− (m/z = 193) is observed with fairly high intensity but [(M − H) − H2 O]− (m/z = 211) from RP is only detected with very low intensity. The dominating sugar cross ring cleavages observed from deprotonated RP and dRP are the 0,2 A and 0,3 A, which lead to m/z = 169 and m/z = 139, respectively. The first channel (0,2 A) is the same as is observed for the most pronounced fragment from the isolated ribose in the gas phase, i.e., m/z = 89. The second channel (0,3 A) constitutes the same bond rupture as the 0,3 X channel observed in the isolated ribose, where this fragmentation also leads to m/z = 89 (see Fig. 11). In RP and dRP, however, due to the high gas phase acidity of the phosphate group, the charge retention is in favour of the formation of the A fragments. Thus 0,2 A and 0,3 A cross ring cleavages are observed in the sugar phosphates compared to 0,2 A and 0,3 X from the isolated d-ribose [31]. Both these fragmentation channels
Fig. 18. Proposed dissociation mechanisms for the C–O and O–P phosphate ester bond cleavage in the sugar phosphates.
are associated with an elimination of 2-C, which explains why both fragment ions appear at the same m/z ratio from RP and dRP, respectively. In addition to the typical sugar fragmentation channels, we also observe the anions PO4 H− 2 (m/z = 97) and PO− 3 (m/z = 79) from both RP and dRP. These constitute phosphate ester bond ruptures at the P–O and the O–C sites, respectively. The phosphate anion PO4 H− 2 is the most intense fragment from both RP and dRP but PO− 3 is also observed with appreciable intensity. The complementary anion, i.e., the charged sugar moiety, is not observed which can be rationalized by the high gas phase acidity of the phosphoric acid group. The proposed fragmentation mechanisms for these two fragments are depicted in Figure 18. The formation of PO4 H− 2 is tentatively assigned to a phosphate ester pyrolysis via a sixmembered intermediate. This fragmentation channel has been proposed for metastable fragmentation of deprotonated oligonucleotides in the gas phase. Ester pyrolysis is also a well known elimination reaction for esters which have an available acidic β proton [56], and plays an important role in thermal degradation of polymers [57]. The PO− 3 anion, on the other hand, is tentatively attributed to a phosphate dissociation. Measurements of DEA to dibutyl phosphate ester show hydrogen abstraction and OH− formation at very low incident electron energies, and an O–C bond rupture at 2 and 4 eV [15]. A more recent study on ribose phosphate shows a DEA resonance near 0 eV leading to the phosphate anion [17]. These results indicate that the presence of the sugar ring opens new dissociation channels that can lead to single strand breaks in DNA by electron attachment at energies lower than 2 eV. 3.5 Metastable fragmentation of deprotonated 2 -deoxynucleoside 5 -monophosphates (dNMPs) The 2 -deoxynucleoside monophosphates (dNMP, N = A, T, G and C) are the combinations of all three building blocks creating the repeat unit in polymeric DNA. A glycosidic bond connects the sugar and the base, and
Eur. Phys. J. D (2012) 66: 13
Page 13 of 20
Fig. 20. Proposed fragmentation mechanism for glycosidic bond rupture in the 2 -deoxynucleotide 5 -monophosphates. A nucleophilic attack from the deprotonated phosphate group to the 4 -C sugar centre induces sugar ring opening and glycosidic bond rupture. This leads to the formation of the fragments [B − H]− and [(M − H) − B]− in a ratio dependent upon the fragments gas phase acidities.
Fig. 19. Metastable decay mass spectra from the 2 deoxy nucleotide 5 -monophosphates; 2 -deoxycytidine 5 -monophosphate (a), 2 -deoxythymidine 5 -monophosphate (b), 2 -deoxyadenosine 5 -monophosphate (c) and 2 deoxyguanosine 5 -monophosphate (d). Fragments in common for the dNMPs are labelled and connected with dotted lines.
a phosphate ester bond connects the phosphate to the sugar through the 3 - or the 5 -oxygen (see Fig. 2). Here we have studied the 5 -monophosphates, dCMP, dTMP, dGMP and dAMP. The PSD spectra of those are displayed in Figure 19. The fragments in common for all the deprotonated − nucleotides are PO− 3 (m/z = 79), PO4 H2 (m/z = 97), − [B − H] (C: m/z = 110, T: m/z = 125, A: m/z = 134, G: m/z = 150), [(M − H) − B − H2 O]− (m/z = 177) and [(M − H) − B]− (m/z = 195). In addition the deprotonated pyrimidine nucleotides eliminate HNCO (dCMP: m/z = 263, dTMP: m/z = 276) and low intensity contribution of NCO− is also observed from dCMP (not shown here). Furthermore, a double water loss leading to [(M − H) − 2H2 O]− is also observed from dCMP (m/z = 294) with fairly high intensity and dGMP shows three additional fragment masses, which are tentatively assigned to [PO4 H + C4 H7 O]− (m/z = 167), [(B − H) + C3 H3 O]− (m/z = 204) and an elimination of H3 C2 O or H3 CN2 (m/z = 303). In general it can be stated that the main fragmentation channels for the nucleotides are the glycosidic bond rupture and the phosphoester bond rupture. The former dominates and leads to the fragments [(M − H) − B]− , [(M − H) − B − H2 O]− and [B − H]− . Due to the higher acidity of the phosphoric ester compared to the sugar moiety and the bases [58], the charge predominantly
remains on the phosphate containing fragments, i.e., [(M − H) − B]− , [(M − H) − B − H2 O]− are the dominating fragments. As the PSD spectra are measured in segments, only a semi-quantitative comparison can be made between individual fragmentation channels within a spectrum. Nonetheless, it is clear that the ratio of the sum of the fragment intensities for [(M − H) − B]− and [(M − H) − B − H2 O]− , to the intensities of [B − H]− are considerably higher for dCMP than the other dNMPs. In the PSD spectra for dCMP this ratio is about 20 but in the others it is about 3. This can be rationalized from the gas phase acidity (GPA) of the nucleobases. The average experimental GPA is in the order C >T ≈ U ≈ A ≈ G (1N − H C, 340; 1N − H T, 333; 1N − H U, 333; 9N − H A, 333; 9N − H G 332; all in kcal/mol [59]). Hence, compared to the other bases, cytosine preferably leaves as the neutral base and the fragments [(M − H) − B]− and [(M − H) − B − H2 O]− dominate over the [B − H]− fragment. The dominance of the glycosidic bond rupture in the PSD spectra of the dNMPs is similar to the observations for the nucleosides, however, in the dNMPs this channel predominantly leads to charge retention on the sugar phosphate site and also additional water loss is observed. We propose that the fragmentation mechanism for the glycosidic bond rupture as well as the additional water loss is similar to the observed mechanism for the glycosidic bond rupture in the nucleosides. In the molecular dynamics simulations of the fragmentation of the nucleosides, a nucleophilic attack was observed from the 2 C–O− and the 3 C– O− to the 1 -C and 4 -C, respectively [33]. This resulted in epoxide formation and rupture of the glycosidic bond. In the dNMPs the deprotonated phosphate group can act as a nucleophile, similar to the hydroxyl groups in the nucleosides. The 5 -position of the phosphate group prohibits base elimination in the dNMPs through an SN 2 attack to the 1 -C. However, as is shown in Figure 20, a nucleophilic attack from the phosphate on the 4 -C of the sugar forms a five membered cyclic phosphate. Similar to the nucleophilic attacks observed for the nucleosides, this leads to an opening of the sugar ring through rupture of the 4 C–O bond, an 1 -C=O carbonyl formation and glycosidic bond rupture. The base is thus eliminated, and the ratio
Page 14 of 20
Eur. Phys. J. D (2012) 66: 13
Fig. 21. Proposed fragmentation mechanism for glycosidic bond rupture and further water loss from the 2 -deoxynucleotide 5 -monophosphate. The initial step is the same as shown in Figure 18, but here the base removes the now acidic 2 -C–H proton to induce E2 elimination of 3 -OH.
between the formation of [B − H]− and [(M − H) − B]− will depend on the proton affinities and gas phase acidities of the departing fragments as well as the reaction rates for the individual channels. Following the nucleobase loss, a further water elimination from the resulting deprotonated sugar phosphate ion [(M − H) − B]− (m/z = 195) also occurs. Due to the carbonyl group on the 1 -C, the 2 -H (now a β hydrogen to the carbonyl) becomes acidic and can thus be eliminated by the departing base to induce E2 elimination dissociating the 3 -C–OH bond and forming the fragment [(M − H) − B − H2 O]− (m/z = 177). This is shown in Figure 21. Again, the low acidity of the nucleobase cytosine results in a higher proportion of 2 -H removal and thus increased ion yield of [(M − H) − B − H2 O]− from dCMP compared to the other dNMPs. The phosphate ester bond ruptures, forming PO4 H− 2 and PO− 3 , have been discussed for the sugar phosphates and we assume the same fragmentation mechanisms to be responsible for the phosphate ester bond ruptures in the dNMPs. However, the phosphoric ester rupture of the P– O bond leading to the monometaphosphate anion PO− 3 dominates in the nucleotides, whereas the O–C bond rupture leading to H2 PO− 4 dominates in the sugar phosphate. Compared to the sugar phosphates, the dNMPs can be stabilized through a hydrogen bond formation between the nucleobase and the phosphate group. In the neutral dNMPs the free OH groups at the phosphoric ester might act as a hydrogen donor and the nucleobase as an acceptor, leading to a partial negative charge at the phosphate group and a partial positive charge at the base. In contrast, in the negatively charged (deprotonated) dNMPs, the nucleobase can act as a hydrogen donor and this arrangement can facilitate a phosphate dissociation and thus preference for the PO− 3 formation. This is also in concord with the different GPAs of the bases, as the ratio of PO− 3 formation to the PO4 H− 2 formation in dCMP, which is the least acidic, is still slightly in favour of the PO4 H− 2 formation whereas the formation of PO− 3 clearly dominates for all the other dNMPs. In summary, we can conclude for the dNMPs that the glycosidic bond rupture and the phosphodiester bond rupture dominate and that the phosphate group has a determining influence on the fragment formation. The sugar cross ring cleavage observed for the isolated ribose and the nucleosides is not observed for the dNMPs, with the exception of dGMP where some low intensity sugar cross
ring cleavage fragments are observed. The base fragmentation observed for the isolated pyrimidine bases and the pyrimidine nucleotides is still apparent through the HNCO loss observed with comparably high intensity from dCMP and also through the low intensity NCO− contribution from the same. However, the influence of the different nature of the bases is mainly through their different proton donor/acceptor properties and less through the chemical difference between the purine versus pyrimidine as observed for the isolated bases and the nucleosides. Due to the thermal instability of the dNMPs and the NMPs no gas phase DEA data has been available on these until very recently. During the final preparation of this manuscript the authors became aware of a gas phase DEA study on 2 -deoxy-cytidine 5 -monophosphate conducted by Kopyra et al. [18]. At 130 ◦ C they could show that only the intact dCMP was desorbed. They found that − the fragments PO− 4 and H2 PO4 CH2 , which both correspond to a backbone rupture, were formed through a low energy resonance close to 0 eV. Also HCOO− , which must be attributed to sugar fragmentation, was observed through this resonance. From these fragments only the PO− 4 fragment results from the same bond rupture as observed in the metastable decay of the deprotonated dCMP. − The fragments H2 PO4 CH− 2 and HCOO both constitute a C–C bond rupture, which we do not observe in our experiments. 3.6 Metastable fragmentation of deprotonated, short single stranded oligonucleotides In addition to the metastable fragmentation of the individual building blocks of DNA and RNA we have studied the metastable fragmentation of a number of hexameric oligonucleotides (ONTs) of different composition [36]. ONTs composed of thymidine nucleotides have previously been reported to show comparably little fragmentation in MALDI. To study the influence of the individual bases and their sequence we have thus chosen all 6 combinations of the hexameric oligonucleotides 5 -d(TTXYTT) (X = Y = G, C and A) as model compounds [36]. In these ONTs the two central nucleotides containing high proton affinity bases, are bracketed by two thymines. Furthermore, to study the role of the acidic hydrogens in the fragmentation of these ONTs we have systematically exchanged them against sodium ions.
Eur. Phys. J. D (2012) 66: 13
Page 15 of 20
a) TTACTT
b4 d4 a5-B5
w3 w2
Fig. 22. Nomenclature for oligonucleotide fragmentation as introduced by McLuckey et al. [61]. Lower case letters signify what bonds are ruptured and whether the charge retention is on the 3 - or the 5 -end. The subscript numbers signify the position of bond rupture within the strand. For the example shown here, a 3 -C–O bond rupture at the first nucleotide from the 5 -end is a w1 fragment. If the charge retention is on the 3 -end, this bond rupture leads to an a3 fragment (three nucleotides from the 3 -end). Correspondingly, an additional base loss from the third nucleotide leads to the formation of an a3 – B3 fragment.
Considerable numbers of studies have been dedicated to ONT fragmentation in MALDI with the bulk of this work being reviewed by Wu and McLuckey in 2004 [60]. It is beyond the scope of this colloquium to revisit these studies and we will, at large, limit the discussion on the ONTs to comparison with the observations for the smaller building blocks. Here we will adhere to a nomenclature suggested by McLuckey et al. [61], that is commonly used for fragmentation of ONTs in MALDI-MS. In Figure 22 this nomenclature is depicted for the example of a single stranded tetrameric ONT. Figure 23 compares the metastable fragmentation of the oligonucleotides 5 -d(TTACTT) and 5 -d(TTCATT). The main fragmentation channels observed are the channels denoted w3 and a4 – B4 . The w3 channel constitutes a rupture of the third 3 -C–O of the backbone counted from the 3 -end, with charge retention on that end. The a4 – B4 channel constitutes a rupture of the fourth 3 -C–O of the backbone, counted from the 5 -end, and the loss of the base on the fourth nucleotide. In this case (a4 – B4 ) the charge retention is on the 5 -fragment. If the higher PA base cytosine is in the third position the w3 fragment dominates and if it is in the fourth position the a4 – B4 fragment dominates. This is also the case for the other ONTs studied with the exception of 5 -d(TTGCTT) and 5 -d(TTCGTT) where the proton affinities of the bases are comparable and the intensity of the w3 and a4 – B4 fragment are also comparable. The observations that the w3 fragment is preferably formed when the higher PA base is in the third position and the a4 – B4 fragment is preferably formed when this base is in the fourth position indicate that the initial step in the backbone cleavage is the loss of a high PA base. Figure 24 compares the PSD spectra of 5 d(TTCATT), where 0–5 protons have progressively been exchanged against sodium ions. The protons that are ex-
d5
-
[M-H]
a4-B4 w3
w2
600
b5
A-loss T-loss C-loss
x10
b) TTCATT
x10
-H2O
a4-B4
x5
800
1000
1200 m/z
1400
1600
1800
Fig. 23. Metastable decay mass spectra from the deprotonated hexameric oligonucleotides 5 -d(TTACTT) and 5 -d(TTCATT). The main fragmentation channels are high PA base loss (A- and C-loss) and further 3 -C–O dissociation at the site of base elimination. This channel leads to the formation of the w3 and a4 – B4 fragments. Other fragmentation channels lead to b and d (or w and y) fragment formation. Due to the symmetric composition of the nucleotides studied, these are indistinguishable from the mass alone. We however assign these to b and d fragments based on the extensive water loss (not observed for w3 ).
changed against the sodium ions are preferably the more acidic ones, i.e., the phosphodiester protons. It is clear from Figure 24 that with increasing numbers of protons exchanged against sodium ions, the w3 and a4 – B4 channels become less pronounced and that after the exchange of three protons against sodium ions they are close to being quantitatively quenched. A similar behaviour is observed for the other backbone cleavage channels (bn and dn ), and this observation was also made for all other 5 -d(TTXYTT) species. The ratio of the mass peak for the high PA base loss to that of the deprotonated parent ion, however, showed a different tendency. Hence, with an increasing number of protons exchanged against sodium ions this ratio increased. It is thus apparent that the availability of an acidic proton at the phosphodiester groups is not a prerequisite for the base loss, but it is necessary for the backbone cleavage to proceed through the w3 and a4 – B4 channels. Hence, if there is one predominating mechanism responsible for the w3 and a4 – B4 channels in these deprotonated ONTs, this is a single base loss, which may be charge controlled and does not rely on proton transfer from the phosphoester groups. The base loss is then followed by a backbone cleavage that can only proceed if acidic phosphoester protons are available. Figure 25 depicts a proposed fragmentation mechanism that is consistent with the observations. Similar to the fragmentation mechanism proposed for the nucleotides a nucleophilic attack from a neighbouring phosphate to the 4 -C site can induce base elimination and sugar ring opening in the ONTs. For neutral elimination of the base, an acidic proton in its vicinity is transferred to the base, and when available, this is most likely its 3 -phosphate proton. This leads to the formation of a 1 -C=O aldehyde at the base
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Fig. 24. Comparison of fragment mass spectra for 5 d(TTCATT) with 0–5 protons exchanged against sodium (Na) ions. See text for discussion.
elimination side and the now deprotonated 3 -phosphate can undergo further ester pyrolysis with the acidic 2 -C–H proton (β-proton with respect to the 1 -aldehyde), to dissociate the 3 -C–O bond and form the observed fragments. If an acidic proton is not available at this phosphate the base elimination can still take place with proton transfer from a neighbouring base, however the further ester pyrolysis is blocked. In summary, we can conclude for the metastable fragmentation of the deprotonated ONTs that glycosidic bond rupture leading to further backbone cleavage at the 3 C–O bond of the respective nucleotide is the dominating fragmentation channel. Also, other fragmentation channels leading to backbone cleavage at the 3 -P–O bond and the 5 -C–O bond (bn and dn fragments) are observed along with additional loss of water. Single base loss is also observed without further fragmentation. The sugar cross ring cleavage observed for the isolated sugar and the ribosephosphate is not observed for the ONTs, and neither is
Eur. Phys. J. D (2012) 66: 13
the base fragmentation that was observed for the isolated pyrimidine bases and the pyrimidine nucleotides. Nonetheless, the influence of the different nature of the bases is still apparent, but this influence is mainly through their different proton donor/acceptor properties and less through the chemical difference between the purine versus pyrimidine bases as was observed for the isolated bases and the nucleosides. To our knowledge no DEA studies have been conducted on ONTs in the gas phase, though an appreciable number of studies have been performed on condensed DNA where the irradiated samples were analyzed with electrophoresis [4,62]. Though these studies are very valuable, they do not reveal any clear details about the fragmentation site or which component is the dominating attachment site. There are however also studies where fragments from ONTs exposed to low energy electrons have been identified by liquid chromatography [63] and other studies where small, neutral fragments desorbing from the exposed, condensed DNA are identified [64–67]. DEA around 10 eV to short ONTs; 5 -d(GCAT), results in both base release (glycosidic bond cleavage) and strand breakage (phosphodiester cleavage) [68]. Experiments with ONTs missing bases, indicate that the electron capture probability of the individual bases determines the damage yield. This suggests that an electron transfer from the nucleobase to the backbone followed by C–O phosphodiester cleavage takes place. Further studies with different nucleotide sequences indicate that efficient coupling between the nucleobases is operative resulting in a pronounced sequence dependence of the observed damage yield [69]. Apparently T containing sequences result in a larger extent of glycosidic bond cleavage (and a higher total damage), whereas the presence of G increases the strand break yield [69,70]. At electron energies around 5 eV desorption of OH− was observed from 40 nucleotide ONTs immobilized on gold surfaces, which was ascribed to a direct electron attachment to the phosphate group followed by OH− abstraction. Further systematic investigations are required to map out all relevant parameters for electron induced DNA damage, including the electron energy, nucleotide length and sequence and their environment.
4 Conclusion In this short colloquium we have summarized recent results on the metastable decay of the individual building blocks of the nucleic acids and their compositions to larger components in order to give a comprehensive picture of how the fragmentation channels observed for the smaller components prevail in their larger compositions. In general we can conclude that the dominating fragmentation channels observed for the individual components prevail when we proceed one step in complexity, for example from the isolated sugars and nucleobases to the respective sugar phosphates and nucleosides. This is also true when moving from the sugar phosphates and nucleosides
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Fig. 25. The proposed fragmentation mechanism forming the fragments a-B and w from deprotonated oligonucleotides. The first step is base elimination similar to what was proposed for the 2 -deoxynucleotide 5 -monophosphates (Fig. 18). For neutral base elimination an acidic phosphate proton is removed with the base. The deprotonated phosphate can undergo phosphate ester pyrolysis with the now acidic 2 -C–H proton to dissociate the 3 -C–O bond and form either of the two fragments a-B or w. The two complementary fragments with respect to proton retention are observed.
to the dNMPs and from the dNMPs to the ONTs. Additionally, new fragmentation channels open up when the individual components are combined. These new channels may dominate the fragmentation of the respective composition, and in turn be the only ones that persist in the next, more complex compositions. For example; the most pronounced cross ring cleavage observed in the isolated sugars is also apparent in the sugar phosphates and in the nucleosides. However, the ester bond rupture dominates in the sugar phosphates and the glycosidic bond rupture and the base fragmentation dominates in the nucleosides. When we take one step further in complexity and combine the sugar phosphates and the nucleosides to form the dNMPs, the most pronounced channels prevail again, i.e., the phosphoester rupture, the glycosidic bond rupture and the base fragmentation. The sugar cross ring cleavage, on the other hand, is not apparent anymore. Furthermore, when the NMPs are combined to the hexameric ONTs the (direct) phosphoester bond rupture is still present (bn and dn fragments), but the glycosidic bond rupture is the dominating channel (although it mainly leads to phosphodiester bond cleavage). For metastable decay it is thus clear that, though the dominating fragmentation channels from the individual components tend to prevail when these components are combined, the dominating fragmentation of the composition may well be through ruptures of the new bonds. These, new channels, may in turn be the only ones that survive when yet another component is added. As this work was largely motivated by the discovery that low energy electrons can resonantly attach to condensed DNA and induce single-, as well as double-strand breaks [4,22], we have compared the metastable fragmentation of the deprotonated components with the fragmentations observed upon DEA. The formation of the negative ions of the high electron affinity fragments NCO and CN, the glycosidic bond rupture of the sugar phosphate, and the nucleosides, as well as the neutral frag-
ments eliminated from ribose are found to be common observations in the PSD studies and previous DEA studies. These are, however, only a few of the fragmentation channels observed upon DEA to DNA components and only a handful of low yield DEA channels in biologically relevant molecules have been unambiguously identified as secondary, metastable fragmentation channels induced by hydrogen abstraction [26,30]. Furthermore, the metastable decay processes studied here are statistical fragmentation processes from the electronic ground state of the respective molecules. In DEA, on the other hand, the fragmentation proceeds from a fairly well defined transient negative ion (resonance) along a repulsive route on the potential energy surface. This resonance may be the anionic ground state (or an excited state) and initial fragmentation channels such as hydrogen abstraction may occasionally lead to further metastable decay. However, in DEA, even very complicated rearrangement reactions are usually observed through fairly narrow resonances and so cannot be seen as a statistical decay. It is thus well possible in DEA, where the formation probability of a given resonance determines the dominating fragmentation path, that the fragmentation characteristics of individual components is better preserved in their compositions than is the case in metastable decay of the deprotonated analogues in their electronic ground state. It is thus clear that, despite significant advances during the last few years, many basic issues remain to be explored to arrive at a more complete understanding of the processes relevant for the current procedures of radiation risk assessment and radiation therapy. In the past, virtually all experiments exploring electron induced DNA damage have been carried out in ultra-high vacuum (UHV) with dried DNA and DNA components. The most important questions that need to be addressed thus concern the role of the environment, i.e. water and dissolved ions, for the interaction between low energy electrons and biomolecules such as the DNA. Not less important are the questions
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relating to how the aqueous environment and ion adduct formation influence the further fate of transient negative ions and intermediate fragmentation products. Furthermore, due to efficient nucleobase stacking, and more generally the interactions between individual nucleotides in a DNA strand, the electronic states (and associated properties such as electron affinity) are largely dependent on the nucleotide sequences. In this context a particular sensitivity for strand-breaks has been suggested for guanine (G) rich single strands [70–72], but also sequences containing predominantly thymine (T) have been shown to be damaged very efficiently [69]. In the cell nucleus DNA is packed within chromatin and exhibits different curvature [73], hence the secondary structure and its association with other biologically relevant molecules such as the proteins is very important for its function, stability and reactivity. Higher-order structures such as G quadruplexes in the telomeres [74] have been shown to have particular electronic properties [75] and play an important role in tumor growth. It is thus important to be able to access information on how higher-order DNA structures alter the susceptibility for low energy electrons. Currently the general assumption is that the most severe consequence of radiation damage, i.e. cancer cell formation, is mainly due to damage caused to the chromosomal DNA in the cell nucleus. The cell lifecycle and its division is however a very complex process and systematic studies that address the influence of radiation on different cell regions and different segments of its lifecycle are important, to verify the significance of the different functions and to define the preferable targets for more detailed studies at the molecular level. The current established methods for analysis of DNA are agarose gel electrophoresis (AGE) and highperformance liquid chromatography (HPLC) [68,69]. However, the detection of single strand breaks in plasmid DNA by AGE is error prone [76] and no information about the damage sites can be obtained. In general the applicability of HPLC and AGE is limited due to the small amount of damaged material resulting from the small penetration depth of low-energy electrons. The analysis of electroninduced damage of ONT sequences longer than tetramers, by HPLC, is thus hampered by the fact that longer sequences generate a large amount of different fragmentation products that then tend to fall below the detection limit [69]. It is therefore a very current need for further development of new methods to understand the role of low energy electrons in the damage caused in living organisms by high-energy radiation. Such methods should preferably enable the production of quasi-free electrons in biologically relevant media, ideally in living cells, but need at the same time to be able to exclude the production of OH radicals and other reactive species. They should also be able to address the mechanism behind the damage in small, intermediate and larger compositions of the DNA components, both when these are isolated in the gas phase and on surfaces and when they are in biological media. Furthermore, to understand the influence of the clinically
Eur. Phys. J. D (2012) 66: 13
very important tertiary structure of DNA, and its aggregation forms with proteins in the cell, it is important to develop methods that can reveal information on the susceptibility of well-defined secondary/tertiary structures and DNA/protein complexes to low energy electron induced damage. Promising experimental approaches have been developed to study electron interactions with complex DNA systems. For DEA measurements in the gas phase laser induced acoustic desorption (LIAD) can be used to transport large and fragile molecules intact into the gas phase [17]. Other promising studies used DNA self-assembled monolayers analyzed by mass spectrometry [77], electron transport measurements [71] and fluorescence spectroscopy [70]. Further emerging methods include the use of DNA nanotechnology to provide welldefined primary and secondary DNA structures that can be systematically varied. After exposure to low-energy electrons, the damage of the structures can then be analyzed by scanning probe microscopy. To approach biologically more relevant systems water must be included, which is experimentally particularly challenging. Controlled addition of water molecules to DNA can, however, be achieved in cluster experiments. The influence of the solvent can also be studied by generating low-energy electrons directly in solution, for instance by using femtosecond UV laser pulses [78], or by irradiation of gold nanoparticles. The use of gold nanoparticles is particularly interesting as they are suggested as potential therapeutics in radiotherapy [79]. A unique tool to study radiation damage on the cellular and sub-cellular level is a microbeam of charged particles, electrons or X-rays that allows for delivery of predetermined doses to specific parts of a cell [80]. The cellular response such as DNA damage and repair and the bystander effect can be studied with that approach to high precision, and specific radiosensitive sites within cells and tissue can be targeted. This work was supported by the Icelandic Centre for Research (RANNIS) and by the University of Iceland Research Fund. HDF acknowledges a Ph.D. grant from the Eimskip University Fund. I.B. acknowledges support for a visit to Reykjav´ık by the COST action P9 (Radiation Damage in Biomolecular Systems, RADAM) and by the European Science Foundation (ESF) program: Electron induced processes at the molecular level (EIPAM). This work was conducted within the framework of the COST Actions CM0601 (ECCL) and MP1002 (NanoIBCT). The authors like to thank Prof. Michael Brunger and Prof. Anne Lafosse for fruitful discussions and their constructive comments during the preparation of this colloquium.
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