J. Phys. Chem. B 2008, 112, 10375–10381
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Chemical Stability and Reactivity of Deprotonated Oligonucleotides (DNA) in the Gas Phase: Protonation and Solvation with Hydrogen Bromide Stefan W. Feil, Greg K. Koyanagi, Janna Anichina, and Diethard K. Bohme* Department of Chemistry, Centre for Research in Mass Spectrometry and Centre for Research in Earth and Space Science, York UniVersity, Toronto, Ontario, Canada M3J 1P3 ReceiVed: May 12, 2008
Selected deprotonated oligodeoxynucleotides generated by electrospray ionization were exposed to a variety of neutral molecules in the gas phase at room temperature in flowing helium gas at 0.35 Torr. Single-stranded [AGTCTG-nH]n- and single- and double-stranded [GCATGC-nH]n- anions were found to be remarkably unreactive with strong oxidants (O3, O2, N2O) and potential intercalators (benzene, pyridine, toluene, and quinoxaline). Hydration also was observed to be inefficient. However, [AGTCTG-nH]n- anions with n ) 2, 3, 4, and 5 were seen to be sequentially protonated and/or hydrobrominated with HBr (but not damaged) and displayed an interesting “end effect” against protonation. Measurements are provided for the rate coefficients of reaction and the efficiencies of protonation. These experimental results point toward the exciting prospect of measuring the intrinsic chemistry of other bare DNA-like anions, including double-stranded oligonucleotide anions in the gas phase at room temperature. 1. Introduction With the availability of electrospray ionization,1 mass spectrometry is able to probe, for the first time, the intrinsic (gas phase) stabilities and chemical properties of multiply charged biological ions, including highly charged deprotonated oligodeoxynucleotides (ODNs). For example, experimental investigations of the gas-phase fragmentation of ODN anions have provided structural information about the primary sequence of a nucleic acid and the nature of its nucleobases.2 However, little is known about the chemical properties of ODN anions in the gas phase. Such information would provide a measure of the intrinsic reactivity of model DNA anions against which reactivity in ViVo can be assessed. Proper transfer of genetic information is well-known to require exact replication and proper transmission of DNA from an ancestor cell to its descendants. Any modifications (mutations) of the corresponding DNA sequence jeopardize proper functioning of the genes or their regulation. Some of the modifications can be repaired, but some cannot, and this results in delayed cellular and molecular effects such as tissue injury, mutagenicity, carcinogenicity, and teratogenicity. How can we understand such effects at the fundamental level? Investigations of the fundamental interactions of fully and partially deprotonated DNA in pristine, solvent-free environments with ambient toxicants could provide important information about the mechanisms of DNA damage from exposure to environmental chemicals. One of the examples of ambient toxicants involves compounds present in primary combustion gases exhibiting high toxic potency, e.g., CO, CO2, O2, HCN, HCl, HBr, and NO2 (smoke products).3 Unlike the collision-induced dissociation of ODNs, which has received a fair amount of attention over the past decade, the chemical reactivity of DNA polyanions is still relatively unknown. The results reported so far include investigations of ion-ion interactions of a hexameric single-stranded ODN * Corresponding author. E-mail:
[email protected].
d(5AAAAAA-H)- with polyamidoamine dendrimers.4 Important structural information relevant to the mechanisms of the gas phase reactivity of DNA models has been obtained with ion mobility measurements combined with molecular modeling.5,6 These studies show that DNA polyanions are able to maintain the helical conformation in the gas phase only if the number of constituent nucleobases exceeds eight; otherwise the helical conformation collapses into a sphere-like structure.5 The importance of cations in the stabilization of multistranded ODNs also has been demonstrated.6 Here we show that DNA polyanions can be injected into a flow reactor and exposed to chemical reaction in the gas phase. We have chosen the hexamer AGTCTG as a model of DNA and studied the reactivity of [AGTCTG-nH]n- (n ) 2-5) anions toward an array of chemical reagents, including the vapor of hydrogen bromide. The neutral reagents selected to interact with the model DNA could be classified into four groups: oxidants (O2, O3, N2O), hydrating agents (H2O, D2O), potential intercalators (benzene, pyridine, toluene, and quinoxaline), and strong acids (HBr). The choice of the reagents was motivated by the plethora of in ViVo processes that are known to involve DNA. Oxidative damage of nucleic acids is known to be the main cause of muta- and carcinogenisis; therefore any studies modeling these processes might contribute to the understanding of DNA damage at the molecular level and accelerate development of novel chemotherapeutics.7 The presence of an optimum number of water molecules defines the conformation of DNA helices and DNA coordination with metal ions and small molecules.8 Understanding the principles of molecular recognition by DNA requires intimate knowledge of the binding modes for ligands and drugs such as groove binding, electrostatic interactions, and intercalation.9 Electrospray ionization tandem mass spectrometry (ESI/MS) has been utilized extensively for the assessment of binding modes and structural characterization of DNA adducts with a vast array of DNA-binding agents, including clinically employed anticancer drugs.10–37 The interaction of model DNA with hydrogen bromide may mimic
10.1021/jp804193u CCC: $40.75 2008 American Chemical Society Published on Web 07/24/2008
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Figure 1. Schematic view of the ESI/SIFT/QqQ instrument.
Figure 2. (Top left) Schematic view of the electrospray sampling region. (Bottom left) ESI spectrum of 20:80 methanol/water solution of AGTCGT (20 µM) at a declustering potential of -30 V. (Right) Structure of [AGTCTG-5H]5-.
the manner in which DNA may be modified upon exposure to combustion mixtures.3 Results reported here comprise the first step of a larger project involving investigations of the influence of charge state, sequence length and composition, and the presence of hydrogen bonding upon the gas-phase reactivity of model DNA polyanions. We also intend to explore the role of multiple metal polycations. Such studies will provide new insight into the intrinsic kinetic and thermodynamic properties of reactions of key biological ions at room temperature. 2. Experimental Section In our laboratory we have coupled an electrospray ion source to a selected-ion flow tube/triple quadrupole (SIFT/ QqQ) mass spectrometer38 that allows the quantitative measurement of gas-phase rate coefficients and product distributions for chemical reactions of charged oligonucleotide anions with gases or vapors of liquids, and we report here the first such results for reactions of deprotonated oligonucleotide dimers and hexamers. A schematic of the
instrument is shown in Figure 1, and a close-up of the electrospray sampling region is provided in Figure 2 together with a sample ESI spectrum. The oligonucleotide anions are introduced into flowing helium gas at 0.35 Torr and allowed to react with neutral reagent gases or vapors added quantitatively downstream. The operation of the instrument has been described recently in some detail, as has the data analysis that provides rate coefficients and product disctributions.38–40 The dideoxynucleotides (DDNs) were electrosprayed from a 100 µM solution in 80/20 water/methanol at a needle voltage of -4500 V. The declustering potential was set to -200 V to maximize the signal of the deprotonated oligonucleotide anion (DDN-H)-. The signal for each data point of a reaction profile was accumulated for 2 min, and the mass range covered included the mass of the fourth cluster (DDNH)(HBr)4-. Similar operating conditions were adopted in the experiments with AGTCTG but with the needle voltage set at -4000 V and the declustering potential at -95 V. Sufficiently intense peaks were obtained for the anion series [AGTCTG-nH]n-, where n ) 2, 3, 4, and 5. The oligonucle-
Stability and Reactivity of DNA in the Gas Phase
J. Phys. Chem. B, Vol. 112, No. 33, 2008 10377 [AGTCTG - nH]5- + O3 f [AGTCTG - nH]O5- + O2
(1)
Another example is the cleavage reaction 2 leading to formation of peroxide and oxide anions. [AGTCTG - nH]5- + O3 f AGTOx3- + CTGOy2(x + y) ) 3
Figure 3. ESI/SIFT/QqQ results for the protonation reaction and clustering of [AGTCTG-5H]5- with HBr molecules in helium buffer gas at 0.35 ( 0.01 Torr and 292 K. Note that measurements of the clusters were taken at different flows. [AGTCTG-5H]5- was derived from a 50 µM solution in 20/80 MeOH/H2O.
SCHEME 1: Summary of the Observed Chemistry
otide GCATGC was electrosprayed with a needle voltage of -4500 V and a declustering potential of -150 V. AGTCTG was purchased from Invitrogen (desalted and cartridge purified). The dimer oligonucleotides AA, GG, TT, and CC and the GCATGC were purchased from ACGT (Toronto), desalted, and cartridge purified. Annealing the single strand in 100 mM ammonium acetate buffer generated the duplex of GCATGC. 3. Results and Discussion 3.1. Remarkable Chemical Stability of Deprotonated Oligonucleotides in the Gas Phase. Somewhat surprisingly, the multiply charged [AGTCTG-nH]n- anions (see Figure 2) were generally found to be quite unreactive in the gas phase. They could not be oxidized with strong oxidants and were not hydrated or protonated with D2O, nor did we observe intercalation with benzene. No reactions, k < 10-13 cm3 molecule-1 s-1, were observed with O2, N2O, O3, D2O, benzene, pyridine, toluene, and quinoxaline for n ) 2, 3, 4, and 5. The extraordinary intrinsic chemical stability of deprotonated oligonucleotides (DNA) has not been demonstrated previously, but might be expected intuitively from Mother Nature that abhors chemical mutations. Nothing appears to be known about the thermodynamics of the oxidation of these polyanions in the gas phase, but stoichiometrically feasible reaction paths are easily envisaged. One obvious example is the O-atom transfer reaction 1 that perhaps could result in oxidation of a nucleobase, but apparently this is kinetically unfavorable.
(2)
Such charge-separation reactions are possibly exothermic, but their thermochemistry is not known. Even if they are exothermic, they could easily be kinetically inhibited by the presence of Coulomb barriers, and this also would account for our failure to observe any ozone, nitrous oxide, or oxygen chemistry. Other searches for intercalation reactions were performed with double-stranded deprotonated GCATGC; we reacted [(GCATGC)2-3H]3- and the single-stranded [GCATGC-2H]2-/doublestranded [(GCATGC)2-4H]4- with benzene, pyridine, toluene, and quinoxaline. No reactions could be observed with any of these molecules, k < 10-12 cm3 molecule-1 s-1, and no adducts were seen in the mass spectra. Apparently these aromatic molecules are poor intercalators in the gas phase; classic intercalators, e.g., ethidium cations, often are positively charged.9 Also, the mechanism of intercalation might be quite different in the gas phase, where the probability of intercalation is influenced by the electrostatic interaction with the charges on the phosphates of the oligonucleotide anion as the intercalator molecule approaches from that in ViVo, where the charges are neutralized by adjacent positively charged histones so that the intercalator can “move around” and so more readily find a gap between adjacent base pairs. Further measurements were made that demonstrate the failure of hydration of single-stranded [GCATGC-2H]2-/double-stranded [(GCATGC)2-4H]4-; there was no observable decay, k < 10-12 cm3 molecule-1 s-1, or adduct formation with D2O. The failure to observe hydration with the various deprotonated oligonucleotides is somewhat curious; perhaps the inefficient hydration can be attributed to weak hydrogen-bonding that may result from the delocalization of the negative charges on the phosphates in these polyanions or the low gas-phase acidity of H2O. 3.2. Protonation of Deprotonated AGTCTG. The intrinsic proton affinities of the different negatively charged phosphate sites of deprotonated oligonucleotides are not known, nor are they known for similar sites as they exist for DNA in ViVo. But the gas-phase acidity of orthophosphoric acid has been reported, 1372 kJ mol-1 > ∆Goacid (H3PO4) > 1331 kJ mol-1,41 and experiments have shown that HBr, ∆Goacid (HBr) ) 1331.8 ( 0.84 kJ mol-1,42 will protonate H2PO4- in the gas phase. So we chose HBr as a reagent and source of protons for our experimental investigations of the protonation of multiply deprotonated AGTCTG. The measured reaction profiles for the observed reactions of HBr with [AGTCTG-5H]5- anion are shown in Figure 3. Scheme 1 presents an overview of the complete chemistry that was identified. Proton transfer and hydrobromination were the only observed reaction channels. There was no evidence for damage by dissociation. Proton transfer from HBr to the [AGTCTG-5H]5- anion (Figure 2) was observed to be rapid, k ) 3.2 × 10-9 cm3 molecule-1 s-1, and also to proceed sequentially to ultimately produce the [AGTCTG-2H]2- anion according to chargeseparation reactions of type 3 with n ) 5-3. [AGTCTG-nH]n- + HBr f [AGTCTG-(n - 1)H](n-1)- + Br-
(3)
Protonation is expected to proceed at the phosphate sites that were originally deprotonated in solution, although the exact
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TABLE 1: Rate Coefficientsa for Reactions of [AGTCTG-nH]n- with HBr in Helium Carrier Gas at (0.35 ( 0.01) Torr and (292 ( 2) K anion
kobsb
kcapc
kobs/kcap
%PTd
[AGTCTG-5H]5[AGTCTG-4H]4[AGTCTG-3H]3[AGTCTG-2H]2-
3.2 2.6 1.9 1.3
3.43 2.75 2.06 1.37
0.93 0.95 0.92 0.95
73 84 20 0
In units of 10-9 cm3 molecules-1 s-1. b Measured effective bimolecular rate coefficient in He at 0.35 Torr and 292 K. The uncertainty is estimated to be ( 30%. c Capture rate coefficient computed according to the algorithm of the modified variation transition-state classical trajectory theory developed by Su and Chesnavich.43 R(HBr) ) 3.61 × 10-24 cm3; µD(HBr) ) 0.82 D.44,45 d The percentage of proton transfer in the presence of HBr addition.
TABLE 2: Computed AM1 Basicities (∆H/kcal mol-1 at 0 K) of the Phosphate Group and the Nucleobase Portion46 dpB-
a
order of sites upon which protonation occurs of course is not known. The observed sequence of protonation indicates that the proton affinities for all these sites are greater than that of Br-, ∆Goacid (AGTCTG) > 1332 kJ mol-1 at 298 K,42 and suggests a similar intrinsic gas-phase acidity limit for DNA. The kinetics for the sequential protonation of [AGTCTGnH]n- is summarized in Table 1. The measured reaction rates increase with increasing charge states from 2- to 5-, but the overall efficiency of reaction, kobs/kcap (where kcap is the computed capture rate coefficient), remains unchanged and equal to 1 within experimental uncertainty. Proton transfer predominates (>70%) with the two highest charge states, drops to 20% for the 3- charge state, and does not occur with the 2- anion. The observation of proton transfer raises interesting questions about the sites of protonation. The five phosphate sites that carry the negative charges can be expected to be the most basic sites. These sites can be expected to have different basicities, even those that are associated with the same nucleobase, because of differences in the molecular environment. Indeed, AM1 calculations by Pan et al.46 for individual deprotonated monodeoxynucleotides (suitably methylated to avoid intramolecular hydrogen bonding) indicate basicities, enthalpies at 0 K, of the phosphate groups in the range from 322 to 327 kcal mol-1 with the four bases T, C, A, and G, and these basicities are from 25 to 47 kcal mol-1 higher than the calculated basicities of the most basic sites on these four nucleobases (see Table 2). In comparison, the acidity (∆H at 0 K) of HBr is 324 kcal mol-1 (1353.5 ( 0.42kJ mol-1).42 So reaction 1 should be nearly thermoneutral or exothermic, and proton transfer from HBr to the negativecharge sites of the phosphate groups should be thermodynamically allowed. But what will be the site and the order in the site of protonation? Perhaps the middle phosphate will be protonated first (either directly or indirectly by intramolecular proton transfer) so that the resulting Coulomb repulsion between the remaining four negative charges will be minimized. The subsequent sites of protonation may then move outward from the central phosphate for similar reasons. The gas-phase conformation should also matter. Should the structure of the multiply deprotonated oligonucleotide be sphere-like rather than helical, any of the nonterminal phosphates could be protonated first. But this is speculation. The interesting failure of the [AGTCTG-2H]2- anion to protonate further was confirmed by the observed absence of Br- in the product ion spectrum of the reaction of selected [AGTCTG-2H]2- anions and can be attributed to an “end effect” that may arise simply from a lower basicity of the
B
site
PA
321.7 323.8
T C
dpA-
325.5
A
dpG-
327.3
G
O4 O2 N3 N1 N3 N7
274.6 289.2 293.6 278.1 301.1 300.3
-
dpT dpC-
SCHEME 2: Illustration of the “End Effect”
terminal phosphates. Alternatively, the formation of a proton bridge between the terminal phosphate anions and the OH substituents of the terminal sugar units could block the potential sites of protonation. This is illustrated in Scheme 2. The hydrogen bond in Scheme 2 could involve an internal phosphate group if a helical conformation is adopted. However, previous ion-mobility experiments mentioned in the Introduction5 indicate that sequences consisting of fewer than eight nucleobases do not maintain the helical conformation in the gas phase but collapse into sphere-like structures. Further insight into the end effect could be gained by derivatizing the hydroxyl groups of the terminal sugars. 3.3. Hydrobromination of Deprotonated AGTCTG. Extensive HBr addition to the oligonucleotide was observed to compete with proton transfer. Eight to eleven molecules of HBr were observed to add to each of the four charge states of the oligonucleotide anions, and this is interpreted as addition to the various basic sites of the nucleobase substituents. But which sites are “in play”? At the He pressure of our experiments (0.35 Torr), HBr addition can be achieved by collisional stabilization (radiative stabilization also cannot be ruled out). This means that addition can compete with proton transfer at the negative charge sites of the phosphate groups according to reaction 4 and can also occur at sufficiently basic sites of the nucleobases according to reaction 5, for example.
PO3-O:- + HBr f [PO3-O:- · · · H+ · · · Br-] f PO3OH + Br- (4a) + He f PO3O-(HBr) + He +
)N: + HBr f )N: · · · H · · · Br |
[
|
(4b)
-
] + He f )N(HBr) + He (5) |
AM1-calculated basicities for the most basic sites of T, C, A, and G are included in Table 2.
Stability and Reactivity of DNA in the Gas Phase
J. Phys. Chem. B, Vol. 112, No. 33, 2008 10379 TABLE 3: Rate Coefficientsa Determined for the Sequential Reactions of Selected Deprotonated Dinucleotides with HBr Molecules in Helium Buffer Gas at 0.35 Torr and 292 K kcollb (TT-H)-
(AA-H)(CC-H)(GG-H)-
7.2 × 7.2 × 10-10 7.3 × 10-10 7.2 × 10-10 10-10
kobs 1.1 × 1.2 × 10-9 1.2 × 10-9 1.0 × 10-9 10-9
k2
k3
5.9 × 10-10 1.1 × 10-9 7.2 × 10-10
1.9 × 10-10
a In units of cm3 molecule-1 s-1. Capture rate coefficient computed according to the algorithm of the modified variation transition-state classical trajectory theory developed by Su and Chesnavich.43 R(HBr) ) 3.61 × 10-24 cm3;44,45 µD(HBr) ) 0.82 D. b
TABLE 4: Experimental Relative Proton Affinities (∆H) of Deoxynucleoside-5′-monophosphates (dpB) and Nucleoside-3′-phosphates (dBp) with B ) A,T, C, and G in kcal mol-1 Measured by the Kinetic Method47,a analyte T A C G
dpB 224.1 ( 1.5 237.4 ( 0.3 236.8 ( 0.5 237.0 ( 0.9
dBp 225.6 ( 0.1 236.4 ( 1.9 234.8 ( 0.5 235.5 ( 0.8
a The uncertainties of the reference values are not included and are less than (2 kcal mol-1.
Figure 4. Structures of deprotonated didexynucleotides.
The data in Figure 3 also do not preclude some occurrence of proton transfer from HBr to the hydrobrominated anions according to reaction 6. [AGTCTG-nH]n-(HBr)m + HBr f [AGTCTG-(n-1)H](n-1)-(HBr)m + Br-
(6)
3.4. Reactions of HBr with Deprotonated AA, GG, TT, and CC. In order to test our proposal for the sites of hydrobromination of [AGTCTG-nH]n-, we performed experiments involving the dinucleotides of each of the four bases present in [AGTCTG-2H]n-, viz., AA, GG, TT, and CC, which
each contain only one phosphate group but different nucleobases only of one kind (see Figure 4). The experimental results displayed in Figure 5 show the extent of hydrobromination that was observed to occur with the four homogeneous dinucleotides at room temperature in the He bath gas at 0.35 Torr. A minor (70%) with n ) 4 and 5, drops to 20% with n ) 3, and does not occur with n ) 2. Failure to protonate the latter was rationalized by a possible “end effect” but can also arise from a lower basicity for n ) 1 and 2. The observation of proton transfer with n ) 3 to 5 indicates basicities for these anions higher than that of Br-, ∆G° > 1332 kJ mol-1. Experiments conducted with four homogeneous DDNs established that hydrobromination can occur at the nucleobases A, C, and G, but not T, in accordance with available computational data on the proton affinities of different sites of protonation. Furthermore, the results point toward the ability to use HBr to count the number of basic sites for longer sequences with different nucleobase composition. This is analogous to the previous use of HI to count basic sites in oligopeptide cations.48 Summarizing the above, we conclude that the reported results point toward the exciting prospect of measuring the intrinsic chemistry of other DNA-like anions, including double-stranded oligonucleotide anions, as well as modeling DNA damage with processes involving common environmental pollutants. Bracketing measurements of proton transfer with a variety of acids should provide a measure of the intrinsic acidities of DNA-like molecules in the absence or presence of metal ions. Acknowledgment. Continued financial support from the Natural Sciences and Engineering Research Council of Canada, the National Research Council, and MDS SCIEX is greatly appreciated. As holder of a Canada Research Chair in Physical Chemistry, D.K.B. thanks the Canada Research Chair Program for their contributions to this research. References and Notes (1) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671. (2) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2004, 237, 197. (3) Levin, B. C. Toxicology 1996, 115, 89. (4) He, M.; McLuckey, S. A. Anal. Chem. 2004, 76, 4189. (5) Gidden, J.; Baker, E. S.; Ferzoco, A.; Bowers, M. T. Int. J. Mass Spectrom. 2005, 240, 183. (6) Baker, E. S.; Bernstein, S. L.; Gabelica, V.; De Paw, E.; Bowers, M. T. Int. J. Mass Spectrom. 2006, 253, 225. (7) Giovangelle, C.; Sun, J. S.; Helene, C. In ComprehensiVe Supramolecular Chemistry; Pergamon Press: Oxford,1996; Vol. 4, p 177. (8) Berman, H. M.; Schneider, B. In Oxford Handbook of Nucleic Acid Structure; Oxford Science Publications: Oxford, 1999; p 2950. (9) Johnson, D. S.; Boger, D. L. In ComprehensiVe Supramolecular Chemistry; Pergamon Press: Oxford, 1996; Vol. 4, p 73. (10) Gale, D. C.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1995, 6, 1154. (11) Triolo, A.; Arcamone, F. M.; Raffaelli, A.; Salvadori, P. J. Mass Spectrom. 1997, 32, 1186. (12) Kapur, A.; Beck, J. L.; Sheil, M. M. Rapid Commun. Mass Spectrom. 1999, 13, 2489.
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JP804193U
