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Low energy electron attachment to N-acetylglycine
Chemical Physics Letters 550 (2012) 47–51
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Low energy electron attachment to N-acetylglycine Janina Kopyra a,⇑, Constanze König-Lehmann b, Eugen Illenberger b a b
Chemistry Department, Siedlce University, 3 Maja 54, 08-110 Siedlce, Poland Institut für Chemie – Physikalische und Theoretische Chemie, Freie Universität Berlin, Takustrasse 3, D-14195 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 17 July 2012 In final form 28 August 2012 Available online 12 September 2012
a b s t r a c t Dissociative electron attachment (DEA) to N-acetylglycine has been studied in the gas phase by means of a crossed beams apparatus. In this Letter we present the results obtained from the target molecule and compare these with the results from its components namely glycine and acetic acid. We find that the number of DEA products is significantly decreased in N-acetylglycine and that the resonance features are notably shifted to lower energies when compared to the individual components. The red shift of the resonances is most likely due to the lower energy of the MOs involved in DEA. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction N-acetylglycine (CH3CONHCH2COOH) is formed by a condensation reaction between acetic acid (CH3COOH) and glycine (NH2CH2COOH, representing the simplest amino acid) thereby releasing a neutral water molecule and forming a peptide bond (Figure 1). In this Letter reactions in the title molecule triggered by resonant attachment of low energy electrons (LEEs) are studied and compared to the components from which the condensation product is formed. The evolution of the properties of individual molecules once they are coupled to larger units is of fundamental interest, in particular when studying biomolecular systems. It has by now widely been accepted that reactions induced by LEEs in biologically relevant molecules are considered as important initial and decisive steps in the molecular description of radiation damage to living tissues [1–3]. This notion is based on the fact that in the course of the interaction of high energy quanta (particles or photons) with biological media (e.g., living cells) copious amounts of secondary electrons are formed having initial energies in the range up to a few tens of eV [4]. Within picoseconds, these ballistic electrons are slowed down by inelastic scattering events (including further ionisation processes) until they become bound as solvated electrons, then as chemically rather inactive species. The particular relevance of LEEs became directly evident by the observation that in plasmid DNA strand breaks can be induced by electrons below the level of ionisation [5,6] and even below the level of electronic excitation (<3 eV). In the latter case, however, only single strand breaks are induced [6]. Since the damage profile versus electron energy showed pronounced resonances it was proposed that resonant electron capture at particular building blocks or sites of the DNA may represent the initial step towards strand ⇑ Corresponding author. E-mail address: [email protected] (J. Kopyra). 0009-2614/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2012.08.058
breaks. In the meantime it has been shown that both, initial electron capture by the DNA bases with subsequent transfer of the excess electron to the backbone and direct electron attachment to the backbone (the sugar and phosphate moiety) contribute to single strand breaks [7]. In the context of radiation damage it has to be noted that not only the damage, which occurs by energy deposition directly in the DNA (direct damage) is relevant but also damage resulting from energy deposition in the vicinity of DNA (indirect damage). Water as the most abundant molecule in a cell generates the reactive OH radical, which can subsequently attack DNA and this kind of indirect damage has been the subject of countless studies [8]. The role of the surrounding proteins with respect to radiation damage is so far not well explored this is however important, as they may protect DNA but at the same time radicals produced from proteins may in turn attack DNA. Amino acids are building blocks for proteins and hence represent vital components of living cells, which have designed particular proteins that specifically bind to single stranded DNA in the course of replication with particular functions [9]. The irradiation of electrons at an energy of 1 eV to an immobilised protein–DNA complex (Escherichia coli bound to a (dT)33 oligomer) demonstrated a significant reduction of single strand breaks as compared to the non complexed (dT)33 oligomer [10]. In light of that, the interaction of LEEs with amino acids and larger units is of direct significance for the investigation of the molecular mechanisms in radiation damage. In studying radiation damage to biomolecular systems one substantial question concerns the evolution of the properties of single molecules once they are covalently coupled to larger units (vide supra). Along that line numerous investigations on electron induced reactions have been performed in the gas phase including the components of the DNA (bases, the sugar and the phosphate moieties) and larger units (base–sugar and phosphate sugar complexes) [1]
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J. Kopyra et al. / Chemical Physics Letters 550 (2012) 47–51
3. Results 3.1. Fragment anions from N-acetylglycine
Figure 1. Molecular structure of N-acetylglycine and the components (acetic acid and glycine) from which it is formed.
up to an entire nucleotide [7]. Apart from single gas phase amino acids [11–17] also peptide bond containing condensation products (glycyl-glycine, alanyl-alanine and glycyl-alanine) have been studied [18,19]. Here we should also mention the recent paper from MALDI experiments, where the metastable decay of the individual, deprotonated components of the nucleic acids and their compositions are discussed in context of DEA studies on those compounds [20]. In the present contribution we study low energy electron driven reactions in N-acetylglycine and compare the results with those from the single components. We find that in N-acetylglycine a series of fragments appear, which are also present in the components but also new decomposition products are observed like the anion due to the loss of a neutral formic acid molecule or a neutral water molecule. The general observation is a significant shift of the resonance features to lower energy in N-acetylglycine with several anionic fragments now appearing right at threshold (zero electron energy) as opposed to the components.
The yield curves of the negative ion fragments observed from N-acetylglycine are presented in Figures 2 and 3. The pronounced resonance profiles indicate that they are the result of (resonant) dissociative electron attachment (DEA). The prominent features are located in the energy range below 3 eV but in some yield curves a comparatively weaker contribution is observed in the energy range around 6 eV. While the low energy contributions can usually be associated to single particle shape resonances (one electron in a normally unoccupied molecular orbital (MO)), the higher energy resonances may be described as two particle core excited resonances (two electrons in normally unoccupied MO) with possibly contributions of higher energy shape resonances. Apparently the ions (M–H)– and OH– are the result of the cleavage of a single bond while the remaining fragments originate from rather complex reactions or reaction sequences of the transient negative ion (TNI) as will be considered in more detail below. (M–H)– is the dominant ion and represents the closed shell anion formed by the loss of a neutral hydrogen atom, which is a well known DEA reaction in organic acids [22]. It is due to hydrogen loss at the OH site as revealed by isotope labelling experiments [23]. Further intense fragments are observed at 71 and 58 u, which can be assigned to (M– HCOOH)–, i.e., the loss of a neutral formic acid molecule (HCOOH) from the TNI and to the CH2COO– anion radical, respectively. Further ions appear at 46 u with the likely stoichiometric composition – H2 CO 2 , and at 26 u (CN / C2 H2 ). A comparatively weaker signal is observed at 99 u, which can be assigned as the ion arising from the loss of a neutral water unit (M–H2O)–. 3.2. Comparison with glycine and acetic acid In both components the loss of a neutral hydrogen atom is the dominant DEA reaction at subexcitation energies, which is also the
2. Experimental The experiments were performed with a crossed electron/ molecular beams apparatus. It consists of a trochoidal electron monochromator, an oven and a quadrupole mass analyzer that are housed in an UHV chamber at a base pressure of 108 mbar. In brief, an electron beam of well defined energy (FWHM 0.2– 0.25 eV, electron current 20 nA) generated from an electron monochromator [21] orthogonally intersects with an effusive molecular beam of gas phase N-acetylglycine. Under ambient temperatures, the sample is solid therefore it was directly deposited into a vessel inside the vacuum system. During the experiments the overall system was heated by two in vacuo halogen bulbs to temperatures of 435–445 K, which is sufficient to generate an effusive molecular beam of the target molecule (pressure of 10–6 mbar as measured with an ionisation gauge mounted at one of the flanges). Negative ions formed in electron-molecule collisions are extracted from a reaction zone towards a quadrupole mass analyzer and detected by a single pulse counting techniques. The intensity of negative ions is recorded as a function of the incident electron energy. The electron energy scale is calibrated using the well known SF 6 signal, which exhibits a sharp peak near 0 eV. N-acetylglycine was obtained from Fluka with a stated purity of P99% and used as delivered.
Figure 2. Yield of the fragment ion due to the loss of a neutral hydrogen atom (M– H) and the ions at 71 u (M–H2CO2) and 46 u H2CO2 (see the text).
J. Kopyra et al. / Chemical Physics Letters 550 (2012) 47–51
49
(ii) the number of fragments is notably lower compared to the components glycine and acetic acid. Both observations can qualitatively be rationalised by the larger size of the condensation product associated with the general trend that the virtual MOs involved in electron attachment are shifted to lower energy with increasing size of the molecule. Accordingly, the larger number of (vibrational) degrees of freedom provides better means for effective randomization of the excess energy thereby suppressing decomposition of the TNI to some degree. In the following we will consider the individual DEA reactions in more detail in relation to the individual components. 4.1. Formation of (M–H)– (loss of a neutral hydrogen atom) Hydrogen loss is an ubiquitous process in organic acids and is due to the hydrogen loss from the O–H [23] site according to the DEA reaction
e þ RCOOH ! RCOOH# ! RCOO þ H
ð1Þ
–#
with RCOOH the transient anion (TNI) formed upon electron attachment. The thermodynamic threshold (the reaction enthalpy, DHo) for a DEA reaction generating two fragments can be expressed as
DHo ¼ DðO HÞ EAðRCOOÞ
Figure 3. Yield of the ions CN/C2 H 2 , OH , CH2COO and the ion due to the loss of a neutral water molecule (M–H2O).
case (as expected) in N-acetylglycine. In electron attachment to acetic acid [24] two further fragments are reported at subexcitation energies, an ion with the stoichiometric composition H2 CO 2 and spurious amounts of HCOO–. The latter is not observed from N-acetylglycine. While in acetic acid the (M–H)– yield peaks at 1.5 eV and that of H2 CO 2 at 0.8 eV, the corresponding ions from N-acetylglycine appear at significantly lower energies. In the case of H2 CO 2 right at threshold (zero eV) and at 0.5 eV for the closed shell anion resulting from hydrogen loss (M–H)–. In glycine a variety of DEA products have been reported previously [11,12], significantly more compared to N-acetylglycine. Apart from the dominant (M–H)– ion further strong signals are observed at 16 u (O–/NH2–), 17 u (OH–), and 26 u (CN–/C2H2–) and on a somewhat lower intensity scale the ions H2CN– (28 u), HCOO– (45 u), H2C2NO (56 u) and an ion at 58 u (H2C2O2–/H2C2NO–). Later on in a high mass resolution experiment [25] it could be shown that the signals at 16 and 26 u are in fact in each case composed of both isobaric fragments, which appear, however, at distinctly different electron energies. Also, NH2– appears at lower energies compared to O– as can be expected from the underlying energetics of the corresponding DEA reactions, and finally CN– at lower energy compared to CCH2–. Accordingly, the study of di-aminoacids glycyl-glycine and glycyl-alanine by a high resolution mass spectrometer [19] demonstrated that the ion at mass 58 consists of C2 H2 O 2 at low energy (around 2 eV) while it is C2H4NO– at higher energy (around 6 eV). 4. Discussion The general in N-acetylglycine is that (i) DEA fragments appear from resonance features located at significantly lower energy and
ð2Þ
with D the bond dissociation enthalpy D(O–H) and EA the electron affinity of the radical (RCOO). In acetic acid the O–H bond dissociation energy is 4.8 eV and the electron affinity of the CH3COO radical 3.4 eV. This yields a thermodynamic threshold of 1.4 eV, which is slightly lower than the experimental peak energy of the RCOO– ion [24]. For the glycine and presently investigated compound the corresponding numbers are not explicitly known but we can assume that the O–H bond dissociation energy should not change to any significant degree for the different acids. But one can expect that the electron affinity of the (M–H) radical increases with the size of the molecule and also depends on its structure. If this is indeed true, then we should observe the bathochromic shift of the peak energy for glycine and even bigger for N-acetylglycine in comparison to acetic acid. This is in fact experimentally confirmed via the observation of the peak position at 1.25 and 1.1 eV for glycine [12] and N-acetylglycine (Figure 2), respectively. H-loss from different organic acids has successfully been described within a one dimensional model using R-matrix theory by initial electron capture into a r⁄(O–H) MO with subsequent direct electronic decomposition along the repulsive (O–H) potential energy curve [22,26]. The one dimensional description uses the anionic potential energy curves of significant energy widths. The calculations reproduce the experimental results very well including the conspicuous features (cusps) on the relative cross section of the (M–H)– yield, which are identified to arise from the vibrational thresholds of the (O–H) vibrations. In the present case the (M–H)– yield also shows distinct structures at spacing of about 0.4 eV, which we accordingly ascribe to cusps arising from the vibrational thresholds. 4.2. Formation of (M–H2CO2)– (71 u) and H2 CO 2 (46 u) The signals at 71 and 46 u (Figure 2) can be assigned to the ions, which are formed from the TNI by the loss of a neutral unit of 46 u and the formation of an ion of 46 u, respectively. From stoichiometry a neutral molecule of 46 u can be due to formation of NO2 or H2CO2. Since the loss of a neutral NO2 molecule or the formation of the ion NO 2 was never observed from the amino acids we assume that a molecule or an ion of the composition H2CO2 is involved. An ion of the composition H2 CO 2 has in fact been previously observed
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J. Kopyra et al. / Chemical Physics Letters 550 (2012) 47–51
from different organic acids like acetic acid [24] and propanoic acid [27] as well as glycyl-glycine [19] and ribose [28]. We hence assume that the ions observed at 71 and 46 u are due to the reactions
e þ M ! ðM H2 CO2 Þ þ H2 CO2
ð3aÞ
e þ M ! ðM H2 CO2 Þ þ H2 CO2
ð3bÞ
with M = C4H7NO3 the target molecule. For reaction (3a) it is very likely that H2CO2 represents the neutral formic acid molecule (HCOOH), which can be formed by a cleavage of the CH2–COOH bond with subsequent hydrogen transfer forming the anion CH3C(O)N(H)CH– (or CH3C(O)N=CH2–). On the other hand, for the reaction (3b) the ion of the composition – H2 CO 2 may not possess the structure of formic acid (HCOOH ) as this negative ion only exists as a short lived scattering state (negative ion resonance) and can hence not bind an extra electron to form a thermodynamical stable anion [29]. From the appearance energy of that ion in acetic acid (0.3 eV) a remarkable stability was predicted (DHof (H2CO2)– 6 790 ± 30 kJmol–1), which is by about 3 eV below its neutral isomeric counterpart HCOOH [24]. From the present compound this ion appears via a resonance directly at threshold but also within a weak resonance feature near 6 eV. It thus appears that an ion of the composition H2 CO 2 has been observed from a number of different targets, its geometric and electronic composition, however, remains to be explored. 4.3. Formation of CN– and/or C2H2– Fragment anions at 26 u appear from two low energy resonant features, one right at threshold (0 eV) the other at 1.8 eV and an additional spurious contribution near 6 eV (Figure 3). In light of the high mass resolution experiment on glycine [25] we can assume that from both low energy resonances CN– is formed while – from the weak feature near 6 eV it is C2 H 2 . The CN feature right at threshold is then characteristic of N-acetylglycine since in glycine it is only formed at higher energies. On the other hand threshold peaks for the CN– formation has been recently reported from derivatives of acetamide namely glycolamide and dibromocyanoacetamide [30]. CN– must be formed via a rather complex reaction ultimately leading to the excision of CN– from the target molecule. Energetically such a reaction is driven by the remarkably high electron affinity of CN (3.8 eV) [31]. However, excision of CN– must be accompanied by the cleavage of five covalent bonds (!) (C–C, N– C, N–H, and two C–H bonds), which is only possible if it is accompanied by the formation of the stable products according to reaction (4a) or (4b)
e ð0 eVÞ þ C4 H7 NO3 ! CN þ CH3 CHO þ H þ HCOOH
ð4aÞ
e ð0 eVÞ þ C4 H7 NO3 ! CN þ CH3 CHO þ H2 þ COOH
ð4bÞ
These reactions would reduce the number of bonds by one thereby producing appreciable stable molecules, i.e. acetaldehyde and formic acid (4a) or acetaldehyde and hydrogen molecule (4b). In addition, a hydrogen atom or COOH neutral product would be formed, respectively. We have then a situation like in the sugars that the molecule, which is kinetically stable, effectively decomposes by just the deposition of an additional charge with almost no energy [28]. 4.4. The loss of a neutral water unit (M–H2O)–, formation of OH– and CH2COO– The loss of a neutral water unit from the target molecule is a comparatively weak reaction and in any case only possible by substantial rearrangement. From the present results, the site at which
the reaction occurs cannot be specified. Effective loss of one or even more neutral water molecules following attachment of very low energy electrons has previously been observed in ribose [28] and other sugar moieties [1]. The OH– ion appears from a broad resonance near 6 eV and additionally near the threshold. The mechanism by which this ion is formed at low energy is not clear as the thermodynamic threshold is at distinctly higher energy. It should be noted that DEA reactions below the thermodynamic threshold can be mediated by vibrational excitation. Due to the peculiarities, e.g., energy dependence of the DEA cross section and lifetime with respect to autodetachment, DEA of such hot band transitions can be remarkably strong [32,33]. However, we cannot exclude that the low energy peak may arise from some impurity or by-product of the decomposition of the target molecule at the hot filament. From the stoichiometric composition the ion at 58 u can be due – to C2 H2 O 2 or C2H4NO . Since in the different di-aminoacids the ion appearing from resonant peak near 2 eV was identified as C2 H2 O 2 [19], we can also assume that from N-acetylglycine it is also – C2 H2 O 2 with the likely structure CH2COO . This would mean a direct cleavage of the N–C bond with concomitant hydrogen transfer generating the closed shell molecule CH3C(O)NH2 (acetamide). In conclusion from the results presented here it can be seen that in N-acetylglycine the DEA resonances are shifted to considerably lower energy with respect to the components from which it is formed (glycine and acetic acid). This leads to DEA reactions already at threshold (0 eV), e.g., the excision of CN–, which occurs via a complex reaction with substantial rearrangement. But the number of DEA products is noticeably suppressed with respect to glycine. Both effects are ascribed to the larger size of the target molecule and accordingly, the lower energy of the MOs involved in DEA and the larger number of vibrational degrees of freedom. Acknowledgments This letter has been supported by the Deutsche Forschungsgemeinschaft (DFG), the Freie Universität Berlin and the Polish Ministry of Science and Higher Education. J.K. gratefully acknowledges support for a visit to Berlin from the EU via the COST action CM0601 (Electron Controlled Chemical Lithography, ECCL). References [1] I. Baccarelli, I. Bald, F.A. Gianturco, E. Illenberger, J. Kopyra, Phys. Rep. 508 (2011) 1. [2] L. Sanche, Eur. Phys. J. 35 (2005) 367. [3] J. Nguyen, Y. Ma, T. Luo, R.G. Bistrow, D.A. Jaffray, Q.-B.- Lu, Proc. Natl. Acad. Sci. USA 108 (2011) 11778. [4] V. Cobut, Y. Fongillo, J.P. Patau, T. Goulet, M.-J. Fraser, J.-P. Jay-Gerin, Radiat. Phys. Chem. 51 (1998) 229. [5] B. Boudaïffa, P. Cloutier, D. Hunting, M.A. Huels, L. Sanche, Science 287 (2000) 1658. [6] F. Martin, P.D. Burrow, Z. Cai, P. Cloutier, D. Hunting, L. Sanche, Phys. Rev. Lett. 93 (2004) 068101. [7] J. Kopyra, Phys. Chem. Chem. Phys. 14 (2012) 8287. [8] C. von Sonntag (Ed.), Free Radical Induced DNA Damage and its Repair – A Chemical Perspective, Springer-Verlag, Berlin, 2006. [9] J.W. Chase, K.R. Williams, Annu. Rev. Biochem. 55 (1986) 103. [10] T. Solomun, T. Skalicky, Chem. Phys. Lett. 453 (2008) 101. [11] S. Gohlke, A. Rosa, F. Brüning, M.A. Huels, E. Illenberger, J. Chem. Phys. 116 (2002) 10164. [12] S. Ptasinska, S. Denifl, A. Abedi, P. Scheier, T.D. Märk, Anal. Bioanal. Chem. 377 (2003) 1115. [13] H. Abdoul-Carime, C. König-Lehmann, J. Kopyra, B. Farizon, M. Farizon, E. Illenberger, Chem. Phys. Lett. 477 (2009) 245. [14] P. Papp, J. Urban, S. Matejcik, M. Stano, O. Ingólfsson, J. Chem. Phys. 125 (2006) 204301. [15] J. Kopyra, Chem. Phys. Lett. 533 (2012) 87. [16] Y.V. Vasil´ev, B.J. Figard, V.G. Voinov, D.F. Barofsky, M.L. Deinzer, J. Am. Chem. Soc. 128 (2006) 5506. [17] J. Kopyra, H. Abdoul-Carime, J. Chem. Phys. 132 (2010) 204302. [18] E. Alizadeh et al., J. Chem. Phys. 134 (2011) 054305. [19] M.V. Muftakhov, P.V. Shchukin, Phys. Chem. Chem. Phys. 13 (2011) 4600.
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H.D. Flosadottir, B. Omarsson, I. Bald, O. Ingolfsson, Eur. Phys. J. 66 (2012) 13. A. Stamatovic, G.J. Schultz, Rev. Sci. Instrum. 41 (1970) 423. V. Vizcaino et al., New J. Phys. 14 (2012) 043017. I. Martin et al., Phys. Chem. Chem. Phys. 7 (2005) 2212. W. Sailer, A. Pelc, M. Probst, J. Limtrakul, P. Scheier, E. Illenberger, T.D. Märk, Chem. Phys. Lett. 378 (2003) 250. [25] A. Mauracher et al., Phys. Chem. Chem. Phys. 9 (2007) 5680. [26] G.A. Gallup, P.D. Burrow, I.I. Fabrikant, Phys. Rev. 79 (2009) 042701. [27] A. Pelc, W. Sailer, P. Scheier, T.D. Märk, E. Illenberger, Chem. Phys. Lett. 392 (2004) 465.
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[28] I. Bald, J. Kopyra, E. Illenberger, Angew. Chem. Int. Ed. 45 (2006) 4851. [29] A. Pelc, W. Sailer, P. Scheier, M. Probst, N.J. Mason, E. Illenberger, T.D. Märk, Chem. Phys. Lett. 361 (2002) 277. [30] C. König-Lehmann, J. Kopyra, I. Da˛bkowska, J. Kocisek, E. Illenberger, Phys. Chem. Chem. Phys. 10 (2008) 6954. [31] The NIST Chemisty WebBook: http://webbook.nist.gov/. [32] I. Hahndorf, E. Illenberger, L. Lehr, J. Manz, Chem. Phys. Lett. 231 (1994) 460. [33] I. Hahndorf, E. Illenberger, Int. J. Mass Spectrom. 167 (168) (1997) 87.
Contents lists available at SciVerse ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Low energy electron attachment to N-acetylglycine Janina Kopyra a,⇑, Constanze König-Lehmann b, Eugen Illenberger b a b
Chemistry Department, Siedlce University, 3 Maja 54, 08-110 Siedlce, Poland Institut für Chemie – Physikalische und Theoretische Chemie, Freie Universität Berlin, Takustrasse 3, D-14195 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 17 July 2012 In final form 28 August 2012 Available online 12 September 2012
a b s t r a c t Dissociative electron attachment (DEA) to N-acetylglycine has been studied in the gas phase by means of a crossed beams apparatus. In this Letter we present the results obtained from the target molecule and compare these with the results from its components namely glycine and acetic acid. We find that the number of DEA products is significantly decreased in N-acetylglycine and that the resonance features are notably shifted to lower energies when compared to the individual components. The red shift of the resonances is most likely due to the lower energy of the MOs involved in DEA. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction N-acetylglycine (CH3CONHCH2COOH) is formed by a condensation reaction between acetic acid (CH3COOH) and glycine (NH2CH2COOH, representing the simplest amino acid) thereby releasing a neutral water molecule and forming a peptide bond (Figure 1). In this Letter reactions in the title molecule triggered by resonant attachment of low energy electrons (LEEs) are studied and compared to the components from which the condensation product is formed. The evolution of the properties of individual molecules once they are coupled to larger units is of fundamental interest, in particular when studying biomolecular systems. It has by now widely been accepted that reactions induced by LEEs in biologically relevant molecules are considered as important initial and decisive steps in the molecular description of radiation damage to living tissues [1–3]. This notion is based on the fact that in the course of the interaction of high energy quanta (particles or photons) with biological media (e.g., living cells) copious amounts of secondary electrons are formed having initial energies in the range up to a few tens of eV [4]. Within picoseconds, these ballistic electrons are slowed down by inelastic scattering events (including further ionisation processes) until they become bound as solvated electrons, then as chemically rather inactive species. The particular relevance of LEEs became directly evident by the observation that in plasmid DNA strand breaks can be induced by electrons below the level of ionisation [5,6] and even below the level of electronic excitation (<3 eV). In the latter case, however, only single strand breaks are induced [6]. Since the damage profile versus electron energy showed pronounced resonances it was proposed that resonant electron capture at particular building blocks or sites of the DNA may represent the initial step towards strand ⇑ Corresponding author. E-mail address: [email protected] (J. Kopyra). 0009-2614/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2012.08.058
breaks. In the meantime it has been shown that both, initial electron capture by the DNA bases with subsequent transfer of the excess electron to the backbone and direct electron attachment to the backbone (the sugar and phosphate moiety) contribute to single strand breaks [7]. In the context of radiation damage it has to be noted that not only the damage, which occurs by energy deposition directly in the DNA (direct damage) is relevant but also damage resulting from energy deposition in the vicinity of DNA (indirect damage). Water as the most abundant molecule in a cell generates the reactive OH radical, which can subsequently attack DNA and this kind of indirect damage has been the subject of countless studies [8]. The role of the surrounding proteins with respect to radiation damage is so far not well explored this is however important, as they may protect DNA but at the same time radicals produced from proteins may in turn attack DNA. Amino acids are building blocks for proteins and hence represent vital components of living cells, which have designed particular proteins that specifically bind to single stranded DNA in the course of replication with particular functions [9]. The irradiation of electrons at an energy of 1 eV to an immobilised protein–DNA complex (Escherichia coli bound to a (dT)33 oligomer) demonstrated a significant reduction of single strand breaks as compared to the non complexed (dT)33 oligomer [10]. In light of that, the interaction of LEEs with amino acids and larger units is of direct significance for the investigation of the molecular mechanisms in radiation damage. In studying radiation damage to biomolecular systems one substantial question concerns the evolution of the properties of single molecules once they are covalently coupled to larger units (vide supra). Along that line numerous investigations on electron induced reactions have been performed in the gas phase including the components of the DNA (bases, the sugar and the phosphate moieties) and larger units (base–sugar and phosphate sugar complexes) [1]
48
J. Kopyra et al. / Chemical Physics Letters 550 (2012) 47–51
3. Results 3.1. Fragment anions from N-acetylglycine
Figure 1. Molecular structure of N-acetylglycine and the components (acetic acid and glycine) from which it is formed.
up to an entire nucleotide [7]. Apart from single gas phase amino acids [11–17] also peptide bond containing condensation products (glycyl-glycine, alanyl-alanine and glycyl-alanine) have been studied [18,19]. Here we should also mention the recent paper from MALDI experiments, where the metastable decay of the individual, deprotonated components of the nucleic acids and their compositions are discussed in context of DEA studies on those compounds [20]. In the present contribution we study low energy electron driven reactions in N-acetylglycine and compare the results with those from the single components. We find that in N-acetylglycine a series of fragments appear, which are also present in the components but also new decomposition products are observed like the anion due to the loss of a neutral formic acid molecule or a neutral water molecule. The general observation is a significant shift of the resonance features to lower energy in N-acetylglycine with several anionic fragments now appearing right at threshold (zero electron energy) as opposed to the components.
The yield curves of the negative ion fragments observed from N-acetylglycine are presented in Figures 2 and 3. The pronounced resonance profiles indicate that they are the result of (resonant) dissociative electron attachment (DEA). The prominent features are located in the energy range below 3 eV but in some yield curves a comparatively weaker contribution is observed in the energy range around 6 eV. While the low energy contributions can usually be associated to single particle shape resonances (one electron in a normally unoccupied molecular orbital (MO)), the higher energy resonances may be described as two particle core excited resonances (two electrons in normally unoccupied MO) with possibly contributions of higher energy shape resonances. Apparently the ions (M–H)– and OH– are the result of the cleavage of a single bond while the remaining fragments originate from rather complex reactions or reaction sequences of the transient negative ion (TNI) as will be considered in more detail below. (M–H)– is the dominant ion and represents the closed shell anion formed by the loss of a neutral hydrogen atom, which is a well known DEA reaction in organic acids [22]. It is due to hydrogen loss at the OH site as revealed by isotope labelling experiments [23]. Further intense fragments are observed at 71 and 58 u, which can be assigned to (M– HCOOH)–, i.e., the loss of a neutral formic acid molecule (HCOOH) from the TNI and to the CH2COO– anion radical, respectively. Further ions appear at 46 u with the likely stoichiometric composition – H2 CO 2 , and at 26 u (CN / C2 H2 ). A comparatively weaker signal is observed at 99 u, which can be assigned as the ion arising from the loss of a neutral water unit (M–H2O)–. 3.2. Comparison with glycine and acetic acid In both components the loss of a neutral hydrogen atom is the dominant DEA reaction at subexcitation energies, which is also the
2. Experimental The experiments were performed with a crossed electron/ molecular beams apparatus. It consists of a trochoidal electron monochromator, an oven and a quadrupole mass analyzer that are housed in an UHV chamber at a base pressure of 108 mbar. In brief, an electron beam of well defined energy (FWHM 0.2– 0.25 eV, electron current 20 nA) generated from an electron monochromator [21] orthogonally intersects with an effusive molecular beam of gas phase N-acetylglycine. Under ambient temperatures, the sample is solid therefore it was directly deposited into a vessel inside the vacuum system. During the experiments the overall system was heated by two in vacuo halogen bulbs to temperatures of 435–445 K, which is sufficient to generate an effusive molecular beam of the target molecule (pressure of 10–6 mbar as measured with an ionisation gauge mounted at one of the flanges). Negative ions formed in electron-molecule collisions are extracted from a reaction zone towards a quadrupole mass analyzer and detected by a single pulse counting techniques. The intensity of negative ions is recorded as a function of the incident electron energy. The electron energy scale is calibrated using the well known SF 6 signal, which exhibits a sharp peak near 0 eV. N-acetylglycine was obtained from Fluka with a stated purity of P99% and used as delivered.
Figure 2. Yield of the fragment ion due to the loss of a neutral hydrogen atom (M– H) and the ions at 71 u (M–H2CO2) and 46 u H2CO2 (see the text).
J. Kopyra et al. / Chemical Physics Letters 550 (2012) 47–51
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(ii) the number of fragments is notably lower compared to the components glycine and acetic acid. Both observations can qualitatively be rationalised by the larger size of the condensation product associated with the general trend that the virtual MOs involved in electron attachment are shifted to lower energy with increasing size of the molecule. Accordingly, the larger number of (vibrational) degrees of freedom provides better means for effective randomization of the excess energy thereby suppressing decomposition of the TNI to some degree. In the following we will consider the individual DEA reactions in more detail in relation to the individual components. 4.1. Formation of (M–H)– (loss of a neutral hydrogen atom) Hydrogen loss is an ubiquitous process in organic acids and is due to the hydrogen loss from the O–H [23] site according to the DEA reaction
e þ RCOOH ! RCOOH# ! RCOO þ H
ð1Þ
–#
with RCOOH the transient anion (TNI) formed upon electron attachment. The thermodynamic threshold (the reaction enthalpy, DHo) for a DEA reaction generating two fragments can be expressed as
DHo ¼ DðO HÞ EAðRCOOÞ
Figure 3. Yield of the ions CN/C2 H 2 , OH , CH2COO and the ion due to the loss of a neutral water molecule (M–H2O).
case (as expected) in N-acetylglycine. In electron attachment to acetic acid [24] two further fragments are reported at subexcitation energies, an ion with the stoichiometric composition H2 CO 2 and spurious amounts of HCOO–. The latter is not observed from N-acetylglycine. While in acetic acid the (M–H)– yield peaks at 1.5 eV and that of H2 CO 2 at 0.8 eV, the corresponding ions from N-acetylglycine appear at significantly lower energies. In the case of H2 CO 2 right at threshold (zero eV) and at 0.5 eV for the closed shell anion resulting from hydrogen loss (M–H)–. In glycine a variety of DEA products have been reported previously [11,12], significantly more compared to N-acetylglycine. Apart from the dominant (M–H)– ion further strong signals are observed at 16 u (O–/NH2–), 17 u (OH–), and 26 u (CN–/C2H2–) and on a somewhat lower intensity scale the ions H2CN– (28 u), HCOO– (45 u), H2C2NO (56 u) and an ion at 58 u (H2C2O2–/H2C2NO–). Later on in a high mass resolution experiment [25] it could be shown that the signals at 16 and 26 u are in fact in each case composed of both isobaric fragments, which appear, however, at distinctly different electron energies. Also, NH2– appears at lower energies compared to O– as can be expected from the underlying energetics of the corresponding DEA reactions, and finally CN– at lower energy compared to CCH2–. Accordingly, the study of di-aminoacids glycyl-glycine and glycyl-alanine by a high resolution mass spectrometer [19] demonstrated that the ion at mass 58 consists of C2 H2 O 2 at low energy (around 2 eV) while it is C2H4NO– at higher energy (around 6 eV). 4. Discussion The general in N-acetylglycine is that (i) DEA fragments appear from resonance features located at significantly lower energy and
ð2Þ
with D the bond dissociation enthalpy D(O–H) and EA the electron affinity of the radical (RCOO). In acetic acid the O–H bond dissociation energy is 4.8 eV and the electron affinity of the CH3COO radical 3.4 eV. This yields a thermodynamic threshold of 1.4 eV, which is slightly lower than the experimental peak energy of the RCOO– ion [24]. For the glycine and presently investigated compound the corresponding numbers are not explicitly known but we can assume that the O–H bond dissociation energy should not change to any significant degree for the different acids. But one can expect that the electron affinity of the (M–H) radical increases with the size of the molecule and also depends on its structure. If this is indeed true, then we should observe the bathochromic shift of the peak energy for glycine and even bigger for N-acetylglycine in comparison to acetic acid. This is in fact experimentally confirmed via the observation of the peak position at 1.25 and 1.1 eV for glycine [12] and N-acetylglycine (Figure 2), respectively. H-loss from different organic acids has successfully been described within a one dimensional model using R-matrix theory by initial electron capture into a r⁄(O–H) MO with subsequent direct electronic decomposition along the repulsive (O–H) potential energy curve [22,26]. The one dimensional description uses the anionic potential energy curves of significant energy widths. The calculations reproduce the experimental results very well including the conspicuous features (cusps) on the relative cross section of the (M–H)– yield, which are identified to arise from the vibrational thresholds of the (O–H) vibrations. In the present case the (M–H)– yield also shows distinct structures at spacing of about 0.4 eV, which we accordingly ascribe to cusps arising from the vibrational thresholds. 4.2. Formation of (M–H2CO2)– (71 u) and H2 CO 2 (46 u) The signals at 71 and 46 u (Figure 2) can be assigned to the ions, which are formed from the TNI by the loss of a neutral unit of 46 u and the formation of an ion of 46 u, respectively. From stoichiometry a neutral molecule of 46 u can be due to formation of NO2 or H2CO2. Since the loss of a neutral NO2 molecule or the formation of the ion NO 2 was never observed from the amino acids we assume that a molecule or an ion of the composition H2CO2 is involved. An ion of the composition H2 CO 2 has in fact been previously observed
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from different organic acids like acetic acid [24] and propanoic acid [27] as well as glycyl-glycine [19] and ribose [28]. We hence assume that the ions observed at 71 and 46 u are due to the reactions
e þ M ! ðM H2 CO2 Þ þ H2 CO2
ð3aÞ
e þ M ! ðM H2 CO2 Þ þ H2 CO2
ð3bÞ
with M = C4H7NO3 the target molecule. For reaction (3a) it is very likely that H2CO2 represents the neutral formic acid molecule (HCOOH), which can be formed by a cleavage of the CH2–COOH bond with subsequent hydrogen transfer forming the anion CH3C(O)N(H)CH– (or CH3C(O)N=CH2–). On the other hand, for the reaction (3b) the ion of the composition – H2 CO 2 may not possess the structure of formic acid (HCOOH ) as this negative ion only exists as a short lived scattering state (negative ion resonance) and can hence not bind an extra electron to form a thermodynamical stable anion [29]. From the appearance energy of that ion in acetic acid (0.3 eV) a remarkable stability was predicted (DHof (H2CO2)– 6 790 ± 30 kJmol–1), which is by about 3 eV below its neutral isomeric counterpart HCOOH [24]. From the present compound this ion appears via a resonance directly at threshold but also within a weak resonance feature near 6 eV. It thus appears that an ion of the composition H2 CO 2 has been observed from a number of different targets, its geometric and electronic composition, however, remains to be explored. 4.3. Formation of CN– and/or C2H2– Fragment anions at 26 u appear from two low energy resonant features, one right at threshold (0 eV) the other at 1.8 eV and an additional spurious contribution near 6 eV (Figure 3). In light of the high mass resolution experiment on glycine [25] we can assume that from both low energy resonances CN– is formed while – from the weak feature near 6 eV it is C2 H 2 . The CN feature right at threshold is then characteristic of N-acetylglycine since in glycine it is only formed at higher energies. On the other hand threshold peaks for the CN– formation has been recently reported from derivatives of acetamide namely glycolamide and dibromocyanoacetamide [30]. CN– must be formed via a rather complex reaction ultimately leading to the excision of CN– from the target molecule. Energetically such a reaction is driven by the remarkably high electron affinity of CN (3.8 eV) [31]. However, excision of CN– must be accompanied by the cleavage of five covalent bonds (!) (C–C, N– C, N–H, and two C–H bonds), which is only possible if it is accompanied by the formation of the stable products according to reaction (4a) or (4b)
e ð0 eVÞ þ C4 H7 NO3 ! CN þ CH3 CHO þ H þ HCOOH
ð4aÞ
e ð0 eVÞ þ C4 H7 NO3 ! CN þ CH3 CHO þ H2 þ COOH
ð4bÞ
These reactions would reduce the number of bonds by one thereby producing appreciable stable molecules, i.e. acetaldehyde and formic acid (4a) or acetaldehyde and hydrogen molecule (4b). In addition, a hydrogen atom or COOH neutral product would be formed, respectively. We have then a situation like in the sugars that the molecule, which is kinetically stable, effectively decomposes by just the deposition of an additional charge with almost no energy [28]. 4.4. The loss of a neutral water unit (M–H2O)–, formation of OH– and CH2COO– The loss of a neutral water unit from the target molecule is a comparatively weak reaction and in any case only possible by substantial rearrangement. From the present results, the site at which
the reaction occurs cannot be specified. Effective loss of one or even more neutral water molecules following attachment of very low energy electrons has previously been observed in ribose [28] and other sugar moieties [1]. The OH– ion appears from a broad resonance near 6 eV and additionally near the threshold. The mechanism by which this ion is formed at low energy is not clear as the thermodynamic threshold is at distinctly higher energy. It should be noted that DEA reactions below the thermodynamic threshold can be mediated by vibrational excitation. Due to the peculiarities, e.g., energy dependence of the DEA cross section and lifetime with respect to autodetachment, DEA of such hot band transitions can be remarkably strong [32,33]. However, we cannot exclude that the low energy peak may arise from some impurity or by-product of the decomposition of the target molecule at the hot filament. From the stoichiometric composition the ion at 58 u can be due – to C2 H2 O 2 or C2H4NO . Since in the different di-aminoacids the ion appearing from resonant peak near 2 eV was identified as C2 H2 O 2 [19], we can also assume that from N-acetylglycine it is also – C2 H2 O 2 with the likely structure CH2COO . This would mean a direct cleavage of the N–C bond with concomitant hydrogen transfer generating the closed shell molecule CH3C(O)NH2 (acetamide). In conclusion from the results presented here it can be seen that in N-acetylglycine the DEA resonances are shifted to considerably lower energy with respect to the components from which it is formed (glycine and acetic acid). This leads to DEA reactions already at threshold (0 eV), e.g., the excision of CN–, which occurs via a complex reaction with substantial rearrangement. But the number of DEA products is noticeably suppressed with respect to glycine. Both effects are ascribed to the larger size of the target molecule and accordingly, the lower energy of the MOs involved in DEA and the larger number of vibrational degrees of freedom. Acknowledgments This letter has been supported by the Deutsche Forschungsgemeinschaft (DFG), the Freie Universität Berlin and the Polish Ministry of Science and Higher Education. J.K. gratefully acknowledges support for a visit to Berlin from the EU via the COST action CM0601 (Electron Controlled Chemical Lithography, ECCL). References [1] I. Baccarelli, I. Bald, F.A. Gianturco, E. Illenberger, J. Kopyra, Phys. Rep. 508 (2011) 1. [2] L. Sanche, Eur. Phys. J. 35 (2005) 367. [3] J. Nguyen, Y. Ma, T. Luo, R.G. Bistrow, D.A. Jaffray, Q.-B.- Lu, Proc. Natl. Acad. Sci. USA 108 (2011) 11778. [4] V. Cobut, Y. Fongillo, J.P. Patau, T. Goulet, M.-J. Fraser, J.-P. Jay-Gerin, Radiat. Phys. Chem. 51 (1998) 229. [5] B. Boudaïffa, P. Cloutier, D. Hunting, M.A. Huels, L. Sanche, Science 287 (2000) 1658. [6] F. Martin, P.D. Burrow, Z. Cai, P. Cloutier, D. Hunting, L. Sanche, Phys. Rev. Lett. 93 (2004) 068101. [7] J. Kopyra, Phys. Chem. Chem. Phys. 14 (2012) 8287. [8] C. von Sonntag (Ed.), Free Radical Induced DNA Damage and its Repair – A Chemical Perspective, Springer-Verlag, Berlin, 2006. [9] J.W. Chase, K.R. Williams, Annu. Rev. Biochem. 55 (1986) 103. [10] T. Solomun, T. Skalicky, Chem. Phys. Lett. 453 (2008) 101. [11] S. Gohlke, A. Rosa, F. Brüning, M.A. Huels, E. Illenberger, J. Chem. Phys. 116 (2002) 10164. [12] S. Ptasinska, S. Denifl, A. Abedi, P. Scheier, T.D. Märk, Anal. Bioanal. Chem. 377 (2003) 1115. [13] H. Abdoul-Carime, C. König-Lehmann, J. Kopyra, B. Farizon, M. Farizon, E. Illenberger, Chem. Phys. Lett. 477 (2009) 245. [14] P. Papp, J. Urban, S. Matejcik, M. Stano, O. Ingólfsson, J. Chem. Phys. 125 (2006) 204301. [15] J. Kopyra, Chem. Phys. Lett. 533 (2012) 87. [16] Y.V. Vasil´ev, B.J. Figard, V.G. Voinov, D.F. Barofsky, M.L. Deinzer, J. Am. Chem. Soc. 128 (2006) 5506. [17] J. Kopyra, H. Abdoul-Carime, J. Chem. Phys. 132 (2010) 204302. [18] E. Alizadeh et al., J. Chem. Phys. 134 (2011) 054305. [19] M.V. Muftakhov, P.V. Shchukin, Phys. Chem. Chem. Phys. 13 (2011) 4600.
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