Effects of Positive and Negative Ionization on Prototropy in Pyrimidine

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A: Spectroscopy, Molecular Structure, and Quantum Chemistry

Effects of Positive and Negative Ionization on Prototropy in Pyrimidine Bases – An Unusual Case of Isocytosine Ewa D. Raczynska, and Mariusz Makowski J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b07539 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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The Journal of Physical Chemistry

Effects of Positive and Negative Ionization on Prototropy in Pyrimidine Bases – An Unusual Case of Isocytosine Ewa D. Raczyńska1,*, Mariusz Makowski2 1

Department of Chemistry, Warsaw University of Life Sciences (SGGW), ul. Nowoursynowska 159c, 02-776 Warszawa, Poland 2

Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland

ABSTRACT: Intramolecular proton-transfers (prototropic conversions) have been studied for the guanine building block isocytosine (iC), and effects of positive ionization, called one-electron oxidation (iC – e → iC+•), and negative ionization, called one-electron reduction (iC + e → iC-•), on tautomeric conversions when proceeding from neutral to ionized isocytosine have been discussed. Although radical cations and radical anions are very short lived species, the ionization effects could be investigated by quantum-chemical methods. Such kind of studies gives some information about the labile protons and the most basic positions in the neutral and radical forms of the tautomeric system. For investigations, the complete isomeric mixture of isocytosine has been considered and calculations performed in two extreme environments, apolar {DFT(B3LYP)/6-311+G(d,p)} and polar {PCM(water)//DFT(B3LYP)/6-311+G(d,p)}. For selected isomers the G4 theory has also been applied. There are no good relations for energetic parameters of neutral and ionized forms. Ionization energies depend on localization of labile protons. Tautomeric equilibria for neutral and ionized isocytosine, favored sites of protonation and deprotonation, and favored structures of protonated and deprotonated forms strongly depend on environment. Acidity of iC+• is close to that of the iC conjugate acid, and basicity of iC-• is close to that of the iC conjugate base. This increase of acid-base properties of charged radicals explains the proton-transfer in ionized pairs of nucleobases. When compared to other pyrimidine bases such as uracil (U) and cytosine (C), which exhibit analogous tautomeric equilibria between nine prototropic tautomers as isocytosine, the tautomeric preferences for iC, iC+•, iC-•, U, U+•, U-•, C, C+•, and C-• are completely different. The differences suggest that acid-base properties of functional groups, their stabilities, and ionization energies play a principal role in protontransfers for pyrimidine bases and influence compositions of tautomeric mixtures.

Keywords: Isocytosine; Effects of ionization; Favored and rare tautomers; Energetic stabilities; Ionization energies; Acid-base properties; DFT, G4, and PCM studies

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INTRODUCTION Prototropy in heterocompounds was already well known,1 when Watson and Crick reported the DNA structure.2 Taking intramolecular proton-transfer equilibria in nucleobases into account, they proposed an interesting mechanism of point mutations,3 developed later by other chemists.4-30 In normal DNA, pyrimidine and purine nucleobases take their canonical forms {Fig. S1 in Supporting Information (SI)} and interact specifically by formation of H-bonds, i.e., cytosine (C) is paired with guanine (G), and thymine (T) is paired with adenine (A). However, in some conditions the canonical forms (C, G, T, or A) can be transformed into their rare forms (C*, G*, T*, or A*), and the natural pairing can be disordered. Nucleobases can be mispaired, e.g., C* can be paired with A, G* can be paired with T, etc. In exceptional cases, when the DNA mismatch repair system does not work well and does not recognize and repair errors during DNA replication, the point mutations in some genes can modify the sequence of amino acids in proteins, enzymes, and receptors.31,32 These changes can affect heredity and aging processes and initiate a development of serious degenerative diseases. In extreme situation, they can drive into organism death. Many factors can influence intramolecular proton-transfers (prototropy) in heterocompounds.33-35 Their effects are not always as simple as could be expected. For this reason, numerous theoretical and experimental investigations have been carried out for free nucleobases and their model compounds, and also for nucleosides, nucleotides, and their dimers modeling pyrimidine-purine base pairs in DNA.4-30 Different factors have been considered such as solvent, other molecule, ion, radical, free or solvated electron, UV-light, gamma-radiation, etc. With development of experimental and theoretical methods, as well as of spectroscopic and computing techniques, the Watson-Crick hypothesis of rare tautomers has been permanently verified and advanced. Owing to importance of this subject, almost each week a piece of information on DNA damages appear in the literature. Investigations for model (or parent) heterocompounds possessing tautomeric group(s) (keto-enol, amide-iminol, enamine-imine, and/or amine-imine) present in nucleobases help to better understand and to explain intramolecular interactions, substituent effects, and


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tautomeric conversions for polyfunctional pyrimidine and purine nucleobases.33-38 In some cases, they even make possible to indicate the pivotal factor(s) that dictate the isomeric structure. For example, investigations on tautomeric equilibria for all possible NH and CH tautomers of imidazole, 2- and 4-aminopyridines, 2- and 4-aminopyrimidines, and purine in the gas phase (apolar environment) gave the possibility to analyze effects of exo and endo groups on tautomeric conversions in adenine.36,37 Additionally, they allowed to formulate a general rule for heterocycles containing the tautomeric amidine group, for which electron delocalization (aromaticity) plays very important role.37,38 For such kind of heterocompounds, the labile proton is transferred between amino and imino N atoms in the amino −C(NH2)=N− and imino −C(=NH)−NH− forms. In particular cases, the ring C atom(s) can also participate in tautomeric enamine-imine conversion(s).36,37,39 O

8OH

N3 4 5 2 1 6

7

H2 N

N

H

H2 N

N

iC2 (N1H-N7H)

OH

HN

H N

N

N

iC6 (N1H-N3H)

H

iC7 (N3H-C5H)

H2N

OH H H

N N

H

H

O

H H

N

N

H

iC5 (N3H-O8H)

O

H

H

N

HN

HN

iC4 (N1H-O8H)

N

iC3 (N3H-N7H))

H

N

H

N

H2 N

H

H

H

O H

O

OH

H

N

HN

H

H

iC1 (N7H-O8H)

H

H

N

H

H

iC8 (C5H-N7H))

H H

N HN

N

H

iC9 (C5H-O8H)

Figure 1. Prototropic tautomers of isocytosine.

Looking for some similarities and differences in prototropy for pyrimidine bases containing at least one tautomeric amide {−C(=O)−NH−} or iminol {−C(OH)=N−} group, isocytosine (iC) has been chosen here and tautomeric conversions studied for its major, minor, and rare tautomers (Fig. 1). This base is a constitutional isomer of the nucleobase C. It contains the same functional groups (amine-imine and amide-iminol) as C but at different positions. Isocytosine is also 2-amino derivative of the other nucleobase uracil (U). Although ACS Paragon Plus Environment

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iC is not present in nucleic acids, the six-membered pyrimidine ring of the nucleobase guanine (G) is structurally identical to that of iC. Recently,40 it has been shown that the excited-state dynamics of iC displays amazing analogy with that of G, indicating that in some cases iC can model physicochemical phenomena occurring in G. The canonical groups in iC possess two labile protons that can move between five conjugated sites (N1, N3, C5, N7, and O8). Consequently, nine prototropic tautomers (iC1iC9 in Fig. 1) are possible for isocytosine, similar to C and U.41-45 Among them iC3 corresponds to the canonical G form and iC1 refers to G* (Fig. S1 in SI). If we take additionally rotational isomerism of the exo −OH group for the hydroxy tautomers iC1, iC4, iC5 and iC9, and geometrical isomerism of the exo =NH group for the imino forms iC4-iC7 and iC9, the isomeric mixture of isocytosine can consist of twenty-one isomers (Fig. S2 in SI). A slightly different situation takes place for G connected with sugar in the DNA or RNA structure. In this case, G possesses two labile protons analogously as iC, but it contains six conjugated sites, five sites in the pyrimidine ring, and additionally, one site (C atom) in the imidazole ring. The imidazole C atom placed between N atoms is conjugated with the canonical groups of the pyrimidine ring. Hence, the tautomeric mixture for G in DNA or RNA (Fig. S3 in SI) can consist of twelve tautomers (twenty four isomers), nine forms similar to those of iC (Fig. 1) and three additional CH tautomers. Favored proton-transfers between heteroatoms (N and O) in G linked with sugar are analogous to those in iC. Note that the fivemembered ring connected with sugar participates very little in isomeric rearrangements in G.617

Only in exceptional cases, e.g., for adiabatically bound valence anion, one of its CH

tautomers with labile proton at the imidazole C atom may become the major form.39 The imidazole ring, connected with sugar in the DNA or RNA structure, is also little engaged in the GC pairing. It mainly acts as substituent and influences acid-base properties of the canonical groups. The sugar residue in the DNA or RNA structure is not conjugated with the canonical groups of G and does not participate in tautomeric conversions. It mainly acts as substituent with polar groups. Quite a different situation takes place for free guanine. It is considerably more complex tautomeric heterosystem than guanine connected with sugar. It contains three labile protons that can move between eleven conjugated sites (all C, N, and O


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atoms). Hence, its tautomeric mixture can consist of forty three tautomers (eighty two isomers), among them twenty eight are CH forms. In this case, isocytosine may be used as reference compound only for a few tautomeric conversions. For neutral isocytosine, a change of tautomeric preferences has already been discovered when proceeding from the gas phase to aqueous solution and in the solid state.41,42,46-72 For investigations, various spectroscopic techniques and quantum-chemical calculations have been applied and the following conclusions reported. The isomer iC3 (Fig. 1), possessing the canonical tautomeric functions like for G in DNA or RNA (Fig. S1 in SI), is favored in aqueous solution, whereas the isomer iC1, corresponding to the rare form G*, predominates in the gas phase or apolar environment. Two tautomeric oxo forms (iC2 and iC3) have been found in the solid state.41,46-48 A change of the isomeric preferences has also been observed for monoprotonated isocytosine when going from apolar to polar medium.40,42,50,51 Effects of ionization (one-electron loss iC – e → iC+• or one-electron gain iC + e → iC-•) have not been analyzed for isocytosine. There is only one document on electron spin resonance (ESR) detection of radical species in a single crystal of isocytosine, irradiated with gamma rays at 77 K.73 However, a few experimental and theoretical reports can be find in the literature for nucleobases. For example, some charged radicals of nucleobases, in particular the C, U and T radical cations, have been investigated in the gas phase by Tureček, Ryzhov and their co-workers, who applied mass spectrometry (MS) techniques, infrared multiple photon

dissociation

photodissociation

(IRMPD)

(UVPD)

spectroscopy

spectroscopy,

in

the

ion-molecule

fingerprint reactions,

region, and

UV/Vis theoretical

calculations.74-76 Bowen and co-workers,39 using photoelectron spectroscopy (PES) and theoretical calculations, investigated the adiabatically bound valence anions of all nucleobases and proved greater stability of CH tautomers than the canonical forms. For the G canonical radical cation, Sevilla and co-workers,77,78 employing ESR and UV-Vis spectroscopy, suggested singly (pH 7-9) and doubly (pH > 11) deprotonated species in frozen aqueous solution. On the other hand, Choi et al.,79,80 using time-resolved resonance Raman spectroscopy combined with pulse radiolysis, demonstrated that the deprotonated G radical


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cation can be rapidly converted by protonation to a new radical cation, its isomer. The experiments of Choi et al. clearly indicated that proton-transfer for ionized nucleobases may be decisive in better understanding the consequences of ionization and DNA damages. The experiments, carried out for selected charged radicals of nucleobases, encouraged us to continue quantum-chemical calculations for pyrimidine bases undertaken about ten years ago.44,45 Generally, quantum-chemical methods complete experimental results.81-83 They give the possibility to study all possible forms of tautomeric systems, whereas experiments lead usually to detection of favored isomers.39,41,46-60,73-80 Signals of rare tautomers are exceptionally difficult to distinguish from the background. Their amounts (< 0.1 %) are too small and cannot be detected experimentally. To obtain the complete picture on protontransfer (tautomeric) equilibria in ionized isocytosine, quantum-chemical methods have been applied for isolated forms in the gas phase that can model apolar environment and for macrosolvated species in polar solvent (water). For our investigations, all nine prototropic tautomers of isocytosine (Fig. 1) have been considered, including conformational and configurational isomerism of the exo −OH and =NH groups, respectively (Fig. S2 in SI). Estimated energetic parameters of iC+• and iC-• isomers could be put together with those previously reported for the neutral iC isomers, investigated at the same levels of theory,42 and effects of ionization analyzed. The ionization effects in the gas phase could be also compared to those previously studied for the ionized pyrimidine nucleobases, cytosine and uracil.43-45 For neutral isocytosine and its charged radicals, deprotonation/protonation reactions could be studied and acid/base properties discussed.

METHODOLOGY For investigations of the ionization effects on prototropy in isocytosine (mixture of twentyone isomers given in Fig. S2 in SI), two extreme environments and two levels of theory have been chosen here as previously described for neutral isocytosine, adenine, and model compounds.36,37,42 For isolated (gaseous) isomers of isocytosine, the density functional theory


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(DFT) method84 with the three-parameter hybrid functional of Becke85 and the non-local correlation functional of Lee, Yang, and Parr86 (B3LYP) has been employed. For solvated (hydrated) isomers of isocytosine, the polarizable continuum model {PCM(water)},87,88 which provides information on medium-polarity effects, has been applied. A 6-311+G(d,p) basis set89 has been used for geometry optimization. The B3LYP functional has been recommended in the literature to study the protontransfer reactions for heterocompounds,90-93 and also used for various tautomeric systems including nucleobases and charged radicals.36,37,43-45,94-103 The PCM method has also been applied for investigations of various forms of the DNA bases.36,37,104-108 The use of the same methods for isocytosine as previously described for other pyrimidine (cytosine and uracil)43-45 and purine (adenine)36,37 bases, gives the possibility to compare their theoretical results and to derive some general conclusions on ionization effects. The DFT and PCM calculations in the present work have been carried out using the Gaussian-03 series of programs.109 The structures of ionized iC+• and iC-• isomers optimized in their ground states at the B3LYP/6-311+G(d,p) and PCM(water)//B3LYP/6-311+G(d,p) levels, their atom coordinates, and electronic energies are given in Table S1 (SI). In Table S2 (SI), the relative energies (∆E for 0 K) of the ionized iC+• and iC-• isomers, calculated in gas phase and aqueous solution, are compared with those found previously for the neutral iC isomers.42 Although all possible twenty-one isomers are stable in both phases for neutral isocytosine, one-electron loss and one-electron gain change the stability of the iC isomers in this way that some of them are not found. For example, one geometrical isomer of the tautomer iC7+• and two rotational isomers of the tautomer iC5-• are unstable at the B3LYP/6-311+G(d,p) and PCM(water)//B3LYP/6311+G(d,p) levels (Table S1 in SI). To estimate the mole fractions of the iC+• and iC-• isomers in the gas phase, vibrational frequencies have been calculated at the same level of theory as that applied to the geometry optimization for isolated species {B3LYP)/6-311+G(d,p)}. The calculations prove that the optimized structures are energy minima. They also give the possibility to estimate the zero-point energy (ZPE) and to calculate thermochemical quantities such as the enthalpy (H), entropy (S), and Gibbs energy (G = H − TS) for 298.15 K. In this way, the relative ∆H, T∆S,


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and ∆G values could be found for tautomeric conversions. The ∆Gs include changes in the electronic energy, zero-point energy (ZPE), and thermal corrections (vibrational, rotational, and translational contributions) to the energy and entropy. Next, the isomeric equilibrium constants K {eq. (1)} between the favored and other isomers could be determined on the basis of the calculated ∆Gs. Finally, the mole fractions y and z of the ionized iC+• and iC-• isomers, respectively, could be found from the estimated Κs similarly as the mole fractions x of the neutral iC isomers {eq. (2)}. The relative thermochemical quantities obtained in the gas phase for the ionized isocytosine isomers are compared with those found previously for the neutral ones in Table S3 (SI). For estimations of the isomeric mole fractions in aqueous solution, the thermal corrections and entropy terms are assumed to be the same in aqueous solution as those in the gas phase.

K = e-∆G/RT

(1)

x (y or z) = Κ/(Σ1nΚ)

(2)

Considering a rapid conversion of one charged radical to the other charged radical (its isomer) by protonation/deprotonation reaction,79,80 it is not always evident for which isomer or for which isomeric mixture experiments have been carried out. When time of measurements is longer than time of isomeric conversions, experimental data can refer to isomeric mixtures, in particular to the mixtures of favored isomers, which may be different for the neutral and ionized forms. When time of experiments is shorter that time of isomeric conversions, it is possible to measure physicochemical quantities for one isomer. These two possibilities of measurements (thermodynamic and kinetic) can be investigated by quantum-chemical methods, and two types of physicochemical quantities (macroscopic and microscopic) estimated for isomeric mixtures and for individual isomers. The microscopic theoretical adiabatic ionization potential IPi {eq. (3)} and the microscopic theoretical adiabatic electron affinity EAi {eq. (4)} could be estimated for each isocytosine isomer in both environments taking the electronic energies (E) of the corresponding iC, iC+• and iC-• isomer possessing analogous constitutions and analogous


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conformations of the tautomeric groups. They are summarized in Table S4 (SI). For the tautomeric mixtures, the macroscopic (average) theoretical adiabatic ionization potential IPm {eq. (5)} and the macroscopic (average) theoretical adiabatic electron affinity EAm {eq. (6)} could be calculated taking the respective electronic energies (E) of the iC, iC+• and iC-• isomers and their mole fractions (x, y, and z, respectively).

IPi = E(optimized radical cation) − E(optimized neutral)

(3)

EAi = E(optimized neutral) − E(optimized radical anion)

(4)

IPm = Σ1nyE(optimized radical cation) − Σ1nxE(optimized neutral)

(5)

EAm = Σ1nxE(optimized neutral) − Σ1nzE(optimized radical anion)

(6)

For deprotonated isocytosine (iC

iCa + H+), six isomers have been selected (Fig.

S4 in SI) and their geometries optimized at the B3LYP/6-311+G(d,p) and PCM(water)// B3LYP/6-311+G(d,p) levels. Their structures, atom coordinates, and electronic energies are given in Table S5 (SI). Their relative energies (∆E for 0 K), calculated in gas phase and aqueous solution, are listed in Table S6 (SI). Vibrational frequencies have been calculated for isolated iCa isomers at the B3LYP)/6-311+G(d,p) level. Their relative ∆H, T∆S and ∆G values, and equilibrium constants (as pK) have been estimated and summarized in Table S7 (SI). The mole fractions (a) of the iCa isomers could be found according to eq. (7) from the estimated Κs {eq. (1)}. For each selected isomer, the microscopic acidity ∆acidHi could be calculated from eq. (8), where H(H+) = Htransl(H+) + pV = 5/2RT = 6.2 kJ mol-1 for 298 K.110115

For calculations, the neutral and deprotonated isocytosine isomers possessing analogous

constitutions and analogous conformations of the functional groups have been taken into account. The microscopic acidities are given in Table S8 (SI). The macroscopic acidity for the tautomeric mixture ∆acidHm could be estimated using eq. (9), where x and a are the mole fractions of the corresponding neutral and deprotonated isomers, respectively, in the gas phase.

a = Κ/(Σ1nΚ)

(7)


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∆acidHi = Hi(anion) + H(H+) − Hi(neutral)

(8)

∆acidHm = Σ1naH(anion) + H(H+) − Σ1nxH(neutral)

(9)

Five isomers of deprotonated iC+• and six isomers of protonated iC-• have been additionally chosen (isomers diC• and piC•, Table S9 in SI) for investigations of deprotonation and protonation reactions of radical cations and radical anions, respectively, to the corresponding neutral radicals. Thermochemical quantities of the isomers diC• and piC•, including those for their acid-base equilibria, have been estimated. Their relative energies (∆E for 0 K) calculated in gas phase {DFT(B3LYP)/6-311+G(d,p)} and aqueous solution {PCM(water)/DFT(B3LYP)/6-311+G(d,p)} are given in Table S10 (SI). The ∆H, T∆S and ∆G values estimated for the isolated neutral radicals are listed in Table S11 (SI). Acidity of the favored radical cation and basicity of the favored radical anion have been determined in similar way as those for neutral isocytosine. For investigations of radicals stability, quantumchemical calculations have been performed for adducts of selected charges radicals with one and eleven water molecules and also with one ammonia, formamidine, and guanidine molecule. Their structures and electronic energies are shown in Table S12 (SI). Additionally, for selected isocytosine isomers of neutral, protonated, deprotonated, and radical forms, calculations have been carried out at 298 K using the G4 theory116 and the Gaussian-09 series of programs.117 Thermochemical quantities have also been calculated at 400 K for two selected neutral iC isomers. The transition states with one imaginary frequency for the intramolecular proton-transfer between the favored iC, iC+• and iC-• isomers have been found at 298 K. The G4-calculated energetic data are given in Table S13 (SI). The HOMO, LUMO, and SOMO have been found for the selected closed and open shell systems and present in Table S14 (SI). The mole fractions for isolated major and minor isomers of various isocytosine forms have been found on the basis of the thermochemical quantities. Acid-base properties of neutral isocytosine and its charged radicals have been estimated at the G4 level according to the same procedure as that applied for the DFT-structures.


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RESULTS AND DISCUSSION Energetic Parameters for Ionized Isocytosine Isomers. Independently on the state of oxidation of isocytosine isomers, variations of the relative energies (∆E for 0 K) estimated for the solvated structures at the PCM(water)//DFT(B3LYP)/6-311+G(d,p) level are almost parallel to those for the gaseous ones found at the DFT(B3LYP)/6-311+G(d,p) level (Fig. 2). This parallelism of energetic parameters observed for iC+• and iC-• isomers (Table S1 in SI) suggests that the mechanisms of positive and negative ionizations do not depend significantly on environment. They seem to be similar in the two extreme phases.

250

Neutrals Radical cations

∆E (water) [kJ/mol]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200

Radical anions

150 100 50 0 0

50

100

150

200

250

∆E (gas) [kJ/mol]

Figure 2. Linear tendencies between ∆Es estimated for solvated and isolated isomers of neutral and ionized isocytosine.

However, some small differences can be distinguished for individual isomers (Table S2 in SI). For example, the orders of isomeric stabilities in aqueous solution are not exactly analogous to that in the gas phase. For the NH-OH and NH-NH tautomers (iC1+•-iC6+•), which can decide about physicochemical and biochemical properties of the tautomeric iC+• mixture, the order of their isomeric stabilities in the gas phase (iC3+• > iC1a+• > iC1b+• > iC4ba+• > iC4bb+• > iC6b+• > iC5ab+• > iC4aa+• > iC6a+• > iC5aa+• > iC2+• > iC4ab+• > iC5bb+• > iC5ba+•) is slightly different than that in aqueous solution (iC3+• > iC6b+• > iC1a+• > iC6a+• > iC4ba+• > iC1b+• > iC4bb+• > iC4aa+• > iC2+• > iC5ab+• > iC4ab+• > iC5aa+• > iC5bb+• > iC5ba+•). For radical anions, the order of their isomeric stabilities in the gas phase (iC8-• > iC7a-• > iC3-• > iC6b-• > iC7b-• > iC6a-• > iC2-• > iC1a-• > iC4ba-• >


11


iC1b-•, etc.) is also slightly different than that in aqueous solution (iC8-• > iC3-• > iC7a-• > iC6b-• > iC6a-• > iC7b-• > iC2-• > iC1a-• > iC1b-• > iC4ba-•, etc.). These variations are mainly a consequence of different intramolecular favorable and unfavorable interactions between functional exo and endo groups and also different intermolecular interactions between polar functional groups and polar solvent which change the internal effects.

(a) Gas Phase {B3LYP/6-311+G(d,p)} 250

Radical cations

∆E (radical-gas)

Radical anions

200 150 100 50 0 0

50

100

150

200

∆E (neutral-gas)

(b) Aqueous Solution {PCM//B3LYP/6-311+G(d,p)} 250

Radical cations Radical anions

E(radical-water)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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200 150 100 50 0 0

50

100

150

200

∆E (neutral-water)

Figure 3. Scatter plots for energetic (∆E in kJ mol-1) parameters estimated in the gas phase (a) and aqueous solution (b) for neutral and ionized isomers of isocytosine.

Remarkable differences exist also between the energetic parameters when their values found for the ionized isocytosine isomers are directly compared to those previously studied for the neutral forms.42 Fig. 3 shows scatter plots for the ∆E values calculated in the gas phase and aqueous solution. Lack of linear relationships between the ∆E values of neutral and ionized isocytosine isomers suggests additionally that energies of ionization are different for individual isomers containing different functional groups. Analogous scatter plots and lack of


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linear relationships can be observed for neutral and ionized isomers of other pyrimidine bases (uracil and cytosine) investigated earlier in the gas phase at the same DFT level.43-45 An additional analysis of the relative energies (Table S2 in SI) calculated for the isomers iC1-iC9, iC1+•-iC9+•, and iC1-•-iC9-• shows interesting consequences of positive and negative ionizations on proton-transfer equilibria. For radical cations, the CH tautomers iC7+•-iC9+• containing one labile proton at C5 display very high relative energies in comparison to the other forms (iC1+•-iC6+•) with the labile protons at O and/or N. This indicates that the CH tautomers are very rare forms and can be neglected in the tautomeric iC+• mixture. Only the amide-iminol and amine-imine conversions are favored for positively ionized isocytosine. The tautomeric iC+• mixture consists mainly of the NH-OH and NH-NH tautomers. Although this general trend is analogous to that observed previously for neutral isocytosine for which the CH tautomers can also be neglected,42 variations of the relative energies for the radical cations are not parallel to those for the neutral isomers. For the radical anions, situation is completely different than that for the radical cations and for the neutral forms. The relative energies of some CH tautomers are smaller than those of the NH-OH and NH-NH ones. In this case, the keto-enol and enamine-imine conversions decide about the tautomeric preferences. Consequently, variations of the ∆Es for the iC1-•iC9-• isomers are not parallel to those for the corresponding iC+• and iC forms. Lack of linear tendencies between the ∆E values of the neutral and ionized isomers indicates that ionization effects strongly depend on the positions of the labile protons in the isocytosine isomers.

Major, Minor, and Rare Tautomers for Ionized Isocytosine. Depending on contribution in the tautomeric mixture, individual tautomers can be considered as major (10% < x < 100%), minor (0.1% < x < 10%), or rare forms (x < 0.1%). The major and minor isomers can be detected experimentally by various spectroscopic techniques, some of them already applied to nucleobases39,74-80 and also to neutral isocytosine.41,46,52,73 However, the rare forms possible in the isomeric mixture can only be investigated theoretically.36,37,39,41-45,54-71,81-83,9496,101-103

Quantum-chemical methods applied to all possible isomers of tautomeric system give

a complete information on tautomeric conversions and on amounts of each isomer in the


13


Page 14 of 44

tautomeric mixture. This kind of information is not accessible on the basis of experimental spectroscopic investigations, particularly for short lived charged radicals. When compared to neutral isocytosine, positive ionization changes the isomeric preferences and the contributions of major, minor, and rare isomers in the isomeric mixture (Table S3 in IS). In the gas phase that models apolar environment, two isomers, iC1a+• with the labile protons at N7 and O8 and iC3+• with the labile protons at N3 and N7 (6.5 and 93.5% at the DFT level, respectively), can be present in significant amounts in the isomeric mixture of positively ionized isocytosine, whereas only one tautomer, iC3+• (100% at the PCM level), can be found in aqueous solution (Scheme 1). The isomer iC3+•, referring to the canonical form, is favored in both environments. However, the isomer iC1a+• is a minor form in the gas phase. Its contribution may be sufficient to model structural changes in ionized base but probably not in polar environment. In aqueous solution, the amount of iC1a+• seems to be lower than 10-5%, and this isomer can be considered as a very rare form. Contributions of other isomers in the isomeric mixture of iC+• are not significant. In both phases, their amounts are lower than 10-5%. They can be neglected as very rare forms. Note that the G4 method, which predicts 9.9% of iC1a+• and 90.1% of iC3+•, reproduces well the DFT results for the favored isomers of positively ionized isocytosine. Moreover, an increase of temperature from 298 to 400 K, tested for the neutral iC1a and iC3 isomers, has no important effect on composition of the iC tautomeric mixture. The percentage contents for iC1a and iC3 are as follows: 97.4 and 2.6 % for 298 K, and 97.2 and 2.8 % for 400 K, respectively. Negative ionization changes dramatically the composition of the isomeric mixture. Very rare isomers for neutral and positively ionized isocytosine become the favored forms for iC-•. In the gas phase, two isomers predominate in the isomeric mixture of negatively ionized isocytosine, iC7a-• with the labile protons at N3 and C5 (0.4 and 0.2% at the DFT and G4 levels, respectively) and iC8-• with the labile protons at N7 and C5 (99.6 and 99.8% at the DFT and G4 levels, respectively), while only one tautomer, iC8-• (100% at the PCM level), in aqueous solution. The DFT and G4 results are analogous for the major and minor radical anions. The percentage contents differ by 0.2%. The isomer iC8-• is favored in both


14

Page 15 of 44

environments. This exceptional situation for iC-• can also occur for negatively ionized guanine (G-•). Indeed, the CH tautomer is very important form for the adiabatically bound valence anions of nucleobases.39 For example, the CH tautomer with one labile proton at C8 predominates for A-•.36,37 For C-• and U-•,43,45 the favored isomers possess one labile proton at C5 like for iC-•. H

O

N H2N

N iC1a

Phase

+.

O H

H

H

H2N

H

N N

.

+

H +

iC3

%

.

%

Gas

6.5 (DFT) 9.9 (G4) Water < 0.01 (PCM)

93.5 (DFT) 90.1 (G4) 100 (PCM) e -

H

O

N H2N

O H

N

O

N H2N

H

N

H

H

H

H2N

H

N N

H

H iC1a Phase Gas Water

iC2

% 79.6 (DFT) 97.4 (G4) < 0.01 (PCM)

iC3

%

%

< 0.1 (DFT) < 0.1 (G4) 2.4 (PCM)

20.4 (DFT) 2.6 (G4) 97.6 (PCM)

e +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O H N

N

H

H

iC7a-

.

Phase Gas

_.

O H H

N

%

0.4 (DFT) 0.2 (G4) Water < 0.01 (PCM)

H H

N H2N

N

.

H

iC8%

99.6 (DFT) 99.8 (G4) 100 (PCM)

Scheme 1. Variations of the tautomeric preferences when going from neutral to ionized isocytosine in the gas phase and aqueous solution.

In the gas phase or apolar environment, each tautomer can be converted into the other one by intramolecular transfer of the labile proton.33-35 The transition states found at the G4 level for the proton-transfer between the major and minor tautomers of neutral (iC1a and iC3), positively (iC3+• and iC1a+•) and negatively (iC8-• and iC7a-•) ionized tautomers of


15


Page 16 of 44

isocytosine are shown in Fig. 4. Each of these transition states possesses one imaginary frequency. Thermal corrections when proceeding from 0 to 298 K and entropy terms have been included for estimation of the Gibbs energy barriers. (a) iC1a → TS → iC3 H

O H

N H2N H

H O

O

N

H

+148

H

N H2N

N

H2N

H

H

N N

H

+9 +•

+•

(b) iC3 → TS → iC1a

+•

H

O N

+.

H2N

N

H H H

O H

N

H2N

+. N

+168

H

N H2N

H

O

+. N

H H

+6 -•

-•

(c) iC8 → TS → iC7a

-•

O N

H N

-.

H H

N

H

H O H

O N H2N

-.

H H

N

H

+155

N

N

-. N

H H H

H +16

Figure 4. Gibbs energy barriers (∆G in kJ mol-1, calculated at the G4 level) for intramolecular proton-transfers in favored neutral (a), positively ionized (b), and negatively ionized (c) isocytosine isomers.

For the favored neutral and positively ionized forms, the labile proton can move from the exo O8 atom to endo N3 atom and vice versa in the amide/iminol group. For the favored radical anions, the labile proton can be transferred from the exo N7 atom to endo N3 atom and


16

Page 17 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


vice versa in the amidine group. The Gibbs energy barriers for neutral and ionized forms of isocytosine are typical for tautomeric conversions in the amide and amidine groups (> 140 kJ mol-1).35,118-121 Positive ionization slightly increases the energy barrier for proton-transfer in comparison to the neutral forms. Other prototropic molecules present in environment usually participate in the intramolecular proton-transfer of tautomeric system and reduce the energy barrier. 35,118-121

Distribution of Charge and Spin Density in Ionized Isocytosine Isomers. As it could be expected the positive charge in iC+• and the negative charge in iC-• are well delocalized in conjugated fragment(s) of the major, minor, and rare isomers. The same is true for the atomic spin density. The mechanisms of one-electron loss and one-electron gain in individual isomers depend on positions of labile protons, constitutions of tautomeric groups, ionization energies, and electron affinities. One electron can be taken from one of π-bonds (C=C, C=N, or C=O) or from one of lone electron pairs of heteroatoms (nN or nO), and next the positive charge and the atomic spin density can be delocalized in the tautomeric radical cation. On the other hand, one electron can be attached by C, N, or O atom, and the negative charge and the atomic spin density can be delocalized in the tautomeric radical anion. Some quantitative information can be derived from the highest occupied molecular orbital (HOMO) in neutral isomers and from the single occupied molecular orbital (SOMO) in charged radicals. In both cases, they are delocalized for favored isomers (Table S14 in SI). The lowest unoccupied molecular orbital (LUMO) in these species is also delocalized but differently than HOMO and SOMO. Distributions of the positive and negative charges on heavy atoms can be estimated taking into account the Mulliken charges calculated for charged radicals together with those for neutral forms. For the major and minor isomers of iC+• and iC-•, changes of the Mulliken charges for the corresponding C, N, and O atoms when going from the neutral to charged forms are shown in Chart 1. The positive charge is delocalized mainly on N1, N3, C5, N7, and O8 in iC1a+• and on N1, C5, N7, and O8 in iC3+•. The negative charge is separated between


17


Page 18 of 44

C6, N1, N7, C4 and O8 in iC7a-•, and between C6, N1, C2, N3, C4, and O8 in iC8a-•. Note that delocalization of the positive and negative charge in the gas phase {B3LYP/6311+G(d,p)} is analogous to that in aqueous solution {PCM(water)//B3LYP/6-311+G(d,p)}.

H

O 0.19 H

0.11 0.02 0.12 N 0.00

+.

0.19

0.09

O 0.08 0.14 0.20

H2 N 0.01 N

0.01 H -0.01 0.11 0.01 0.13 0.14 0.18

H

H

0.03 N -0.01 -0.03 0.05

H2N 0.03 0.10

0.07 -0.01

.

iC1a+

N 0.14 0.20

0.18 0.12

0.01 H -0.01

iC3+

.

-0.10

O -0.07 H

0.04 0.00

N

0.01 0.08 -0.05 -0.08

-0.20 -0.24 N -0.02 N -0.27 0.06 -0.29 -0.08 H -0.16

iC7a

-

.

-0.14 -0.02 O

H H H

-0.07 -0.11 N

_.

0.05 0.07 -0.04 -0.04

H2N -0.07 N 0.03 -0.02

-0.08

-0.05 -0.11

iC8

-0.27 -0.24

-

H H H

.

Chart 1. Variations of the Mulliken charges on heavy atoms in the major and minor isomers of iC+• and iC-• estimated in the gas phase (normal style values) and aqueous solution (italic style values).

The spin density exists on all atoms in the major, minor, and rare isomers of iC+• and iC-•. Its distribution is analogous in both phases. For example, the most important amount is delocalized on the C5 atom in the favored radical cations iC1a+• and iC3+• (Chart 2). Its high concentration is also present on N1 and N7 in iC1a+• and on N1, N7, and O8 in iC3+•. In the other rare isomers, high amount of the spin density is carried by different atoms, i.e., C5, N1 and N7 in the amino-hydroxy isomer iC1b+•; C5, O8 and N1 in the amino-oxo tautomer iC2+•; N7, C5, N1 and N3 in all isomers of the imino-hydroxy forms iC4+• and iC5+• and also in all isomers of the imino-oxo tautomer iC6+•; mainly N7 in the imino-oxo isomer iC7a+• and in all isomers of the imino-hydroxy form iC9+•; N1, O8, C5 and N3 in the amino-oxo tautomer iC8+•. Different distribution of the spin density on C, N, and O confirms that the mechanism of one-electron loss for the individual iC+• isomers depends on functional groups and ionization energies (vide infra). Analogous distribution of spin density exists for the


18

Page 19 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


selected radical cation iC3+• in its adducts with water, ammonia, formamidine and guanidine (see structures in Table S12 in SI).

H

O 0.19 H

0.05 -0.05 0.07 N -0.06

0.61 0.60

H2N -0.04 N

-0.15 -0.15 0.31 -0.06 0.29 0.34 0.28

H

H H2N

0.11 0.16

.

0.00

.

0.02 -0.04

0.64 0.65

N -0.14 -0.15 0.32 0.33

iC3+

H

.

0.05 0.03 O

O 0.00

iC7a-

H

0.00 N -0.12 -0.01 -0.08

iC1a+

-0.01 0.06 H -0.01 -0.03 -0.05 N 0.01 0.24 0.22 N 0.04 N 0.64 0.05 0.72 0.05 H 0.02

+.

0.25

0.04

O 0.03

H H H

0.03 0.04 0.00 N 0.07

H2 N 0.05 0.08

0.18 0.21

_. -0.09 -0.09

N

0.74 0.73

-0.06 -0.08

iC8

-

H H H

.

Chart 2. Distribution of the unpaired spin density on heavy atoms in the major and minor isomers of iC+• and iC-• in the gas phase (normal style values) and aqueous solution (italic style values).

All iC-• isomers studied here can be considered as covalent charged radicals in their ground states. For the favored isomers iC7a-• and iC8a-•, the spin density is mainly carried by C6 (Chart 2). However, its high amount is also on N7 in iC7a-• and on C2 in iC8a-•. For the other rare isomers, the highest amounts of the spin density exist on atoms of the pyrimidine ring, mainly on C6. For isomers of iC1-•-iC6-•, important concentration is also carried by N3 and C4. A participation of the exo N7 atom in spin density delocalization is only significant in all isomers of the imino CH tautomers iC7-• and iC9-•. The exo O atom contributes marginally in this delocalization for all iC-• isomers. Analogous distribution of spin density is observed for the selected radical anion iC8-• in its adducts with water, ammonia, and formamidine (see structures in Table S12 in SI). Unfortunately, there are no experimental and theoretical reports on the spin density distribution for the iC+• and iC-• isomers, and no comparison can be made. Nevertheless, for the C, T, and U radicals and for their Me derivatives the DFT-estimated spin densities agree well with the experimental FT EPR results.122


19


Page 20 of 44

Ionization Potentials and Electron Affinities for Individual Isocytosine Isomers and for Its Tautomeric Mixture. The positive (iC – e → iC+•) and negative ionization (iC + e → iC-•) processes can be described by their energetic quantities such as the ionization potential (IP), called also ionization energy (IE), and the electron affinity (EA), respectively. In the literature, there are numerous experimental IPs and EAs for organic compounds including nucleobases. Most of them are compiled in the NIST Chemistry WebBook.110 Unfortunately, there are no experimental IPs and EAs for isocytosine. Nevertheless, they can be estimated using quantum-chemical methods.97-100,104 For all possible major, minor, and rare isomers of isocytosine, the microscopic theoretical adiabatic ionization potentials (IPi) and the microscopic theoretical adiabatic electron affinities (EAi) could be calculated in both environments using equations (3) and (4), respectively. These microscopic quantities may help to understand some complex processes at a molecular level that may cause changes in nucleic acids. They may also be useful in explanation of some processes that may change structural, physicochemical and biological properties of other tautomeric natural and model systems containing the amide/iminol and/or amidine groups. For IPi and EAi calculations, the electronic energies (E) of the corresponding iC, iC+• and iC-• tautomers having analogous conformation and/or configuration of the exo functional groups have been taken. The IPi and EAi values (Table S4 in SI) estimated for isocytosine isomers vary from 7.9 to 9.4 eV and from -0.6 to 0.9 eV in the gas phase, and from 5.6 to 7.0 eV and from 1.5 to 2.8 eV in aqueous solution, respectively. These variations in the IPi and EAi values confirm their dependence on functional groups which are different in individual tautomers. Model compounds such as aniline, phenol, and pyrimidine, containing the same functional groups as those present in isocytosine isomers, have also different experimental IPs in the gas phase (7.72, 8.49, and 9.33 eV, respectively).110


20

Page 21 of 44

(a) Microscopic IPi

IP i(water)

8 7 6 5 4 7,5

8

8,5

9

9,5

10

IPi(gas)

(b) Microscopic EAi 3 2,5

EAi(water)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 1,5 1 -1

-0,5

0

0,5

1

EAi(gas)

Figure 5. Linear tendencies between microscopic IPi (a) and EAi (b) values (in eV) estimated for solvated and gaseous isocytosine isomers.

Variations of the microscopic IPi and EAi values for isocytosine isomers in the gas phase are almost similar to those in aqueous solution (Fig. 5). In both phases, the lowest IPi values are found for the imino-hydroxy isomers of the tautomers iC4 (N1H-O8H) and iC5 (N3H-O8H), and the largest ones for the imino-oxo isomers of the tautomer iC7 (N3H-C5H). On the other hand, the amino-hydroxy isomers of the tautomer iC1 (N7H-O8H) possess the lowest EAi values, and the amino-oxo tautomer iC8 (N8H-C5H) has the largest ones. These linear tendencies confirm some analogies in ionization processes for isocytosine isomers in these two extreme environments. If we assume that isocytosine tautomers convert rapidly and the time of measurements of the adiabatic IP and EA is longer than the time of tautomeric conversions, one may determine ionization parameters for the tautomeric mixture. These quantities refer to the ionization processes from the favored neutral form(s) to the favored ionized form(s). For such exceptional case, the macroscopic (average) theoretical adiabatic ionization potential (IPm) and the macroscopic (average) theoretical adiabatic electron affinity (EAm) can be estimated.


21


Page 22 of 44

For isocytosine, the major and minor isomers (Scheme 1) could be considered in the IPm and EAm estimations according to equations (5) and (6), respectively. The rare isomers (x, y, and z < 0.1%) could be neglected. The adiabatic IPm and EAm values for isocytosine, calculated in the

gas

phase

{B3LYP/6-311+G(d,p)}

and

in

aqueous

solution

{PCM(water)//B3LYP/6311+G(d,p)}, are as follows: IPm = 8.32 and 6.04 eV and EAm = 0.07 and 2.00 eV, respectively. They are close to the ionization processes for the major isomers in the gas phase, iC1a – e → iC3+• (8.33 eV) and iC1a + e → iC8-• (0.06 eV), and in aqueous solution, iC3 – e → iC3+• (6.05 eV) and iC3 + e → iC8-• (2.00 eV). Using the G4 method, the following values are found in the gas phase: 8.43 (for iC1a – e → iC3+•) and 0.11 eV (for iC1a + e → iC8-•), respectively. Note that they are of the same order of magnitude as the experimental data

reported for gaseous cytosine {IPexp = 8.45 eV123 and EAexp = 0.085 eV for the amino-hydroxy form124}. The IP and EA values for isocytosine change in aqueous solution by ca. 2 eV. An analogous solvation effects have been documented for nucleobases.104,125-127

Acidity of Individual Isocytosine Isomers and Tautomeric Mixture. Neutral isocytosine isomers exhibit amphiprotic properties similar to other isomers of tautomeric systems. They contain both the basic groups that can attach a proton and the acidic groups that can lose a proton. Although there are four heteroatoms in neutral iC isomers, only two of them, the imino N atom and the carbonyl O atom, can be considered as potential sites of protonation. Due to n-π conjugation in the amidine, amide, or iminol groups (:X−C=Y ↔ X+=C−Y:-, where X and Y are NH2 and N, NH and O, or OH and N, respectively), the amino N atom and the hydroxy O atom are the last protonated sites (Chart 3).111,112 The ring C atom can also be neglected in the protonation reaction.128 On the other hand, all NH and OH groups in the neutral iC isomers can be deprotonated. Deprotonation of the CH group is less probable due to smaller electronegativity of C than N and O. All potential sites of protonation and all potential sites of deprotonation in three selected iC isomers (iC1a, iC2, and iC3) are indicated by red arrows in Chart 3.


22

Page 23 of 44

H

O

. . H2N

N

H

H2N

N

O H

H

H

H2N

. .

N

. .

N

. .

. .

H

N

. : .

:

O

. .:

.: .

.: .

(a) Protonation Sites :

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H

N

H

H iC1a

iC2

iC3

.. :O

.. :O

(b) Deprotonation Sites H

..

O: H

:N H

..

N

N ..

H iC1a

H

H

:N H

..

..

H

H

N

N

iC2

H

H H

..

N

H

N: N ..

H

H

iC3

Chart 3. Potential protonation (a) and deprotonation (b) sites in selected isocytosine isomers.

Unfortunately, experimental proton acidity and proton basicity parameters in the gas phase have not been reported for isocytosine.110-112 There are only some documents on its acid-base properties in aqueous solution, and on its experimental pKas for deprotonation of the isocytosine monocation to its neutral form (iCH+ deprotonation of neutral isocytosine to its monoanion (iC

iC + H+, pKa 4.0) and for iCa + H+, pKa 9.6).41,50,51 The

experimental results have been confirmed by theoretical calculations.41 In our previous paper on the favored and rare isomers of neutral isocytosine,42 the macroscopic proton affinity (PAm 937 kJ mol-1) has been estimated in the gas phase at the B3LYP/6-311+G(d,p) level as the enthalpy change of the reaction iCH+

iC + H+ for the

tautomeric system. The G4 method predicts slightly smaller PAm (933.7 kJ mol-1) than the DFT method. It has also been shown by the DFT computations that the endo N1 atom, conjugated with the exo −NH2 group placed at 2-position, is the favored site of protonation in the major iC1a isomer. For comparison, the endo N3 atom, also conjugated with the exo −NH2 group but at 4-position, is protonated in the nucleobase cytosine.129 In both cases, isocytosine and cytosine, the n-π conjugated amidine group (H2N−C=N−) dictates the basicity of the system,111,112 while the amide group (O=C−NH−) decides about basicity of the nucleobase uracil.130 In aqueous solution, both N1 in iC3 or N3 in iC2 can be protonated


23


Page 24 of 44

yielding the common monoprotonated form iC2/3H+, which predominates for the isocytosine monocation (Scheme 2).41,42,50,51 In the gas phase, this monocation is a rare form, and the iC1a/2H+ isomer is favored.42 The most reasonable structures (iCa1-iCa4) for the isocytosine anion (Fig. S4 in SI) are a consequence of deprotonation reaction of the endo >NH, exo −NH2, or exo −OH groups in the selected tautomers of neutral isocytosine given in Chart 3. As could be expected,41,50,51 deprotonation of both N1H in iC2 or N3H in iC3 leads to the common anion iCa1, which is favored (100%) in the tautomeric mixture of deprotonated isocytosine in aqueous solution (Table S6 in SI). However, in the gas phase (Scheme 2) two anionic isomers iCa1 and iCa2a contribute significantly in the anionic mixture (32.5 and 67.5% at the DFT level, and 89.6 and 10.4% at the G4 level, respectively). They can be formed from the major and minor isomers of neutral isocytosine by deprotonation of O8H in iC1a and of N3H or N7H in iC3. The other deprotonated isomers can be neglected for the isocytosine anion. Their DFT-computed relative Gibbs energies are larger than 20 kJ mol-1 (Table S7 in SI). Proton acidity of isocytosine in the gas phase, defined as the enthalpy change of the deprotonation reaction iC

iCa + H+, can be estimated in the microscopic or macroscopic

scale. The microscopic acidity (∆acidHi) corresponds to the deprotonation of the N1H, N3H, N7H, or O8H site in the selected iC isomers, whereas the macroscopic acidity (∆acidHm) refers to the deprotonation reaction from the mixture of major and minor neutral iC isomers to the mixture of major and minor anionic iCa isomers. In the other words, microscopic quantity describes acidity of individual isomer which exists at sufficient time to be separated and analyzed, whereas macroscopic quantity is determined for the mixture of isomers being in equilibrium, i.e., when prototropy and isomerization processes for neutral and deprotonated forms are in equilibrium with intermolecular proton-transfer during acidity measurements.


24

Page 25 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H

O

O H

H

N H2N

+ N

H2N

H

N

H

H

H iC1a/2H+ Phase

H

N

+

iC2/3H+

%

%

Gas

100 (DFT) 100 (G4) Water 0.3 (PCM)

< 0.1 (DFT) < 0.1 (G4) 99.7 (PCM) +

+H

+

-H

iC1a

iC2

iC3

+

+H

+

-H

O H

O H

N

N

_

N

N

H

H2N

H _ N

H

H iCa1

iCa2a Phase

%

Gas

67.5 (DFT) 10.4 (G4) Water < 0.01 (PCM)

% 32.5 (DFT) 89.6 (G4) 100 (PCM)

Scheme 2. Isomeric preferences for protonated and deprotonated isocytosine in the gas phase and aqueous solution. DFT and PCM data for the protonated isomers taken from ref. 42.

The microscopic acidities, calculated at the DFT and G4 levels according to eq. (8), give information on the favored site of deprotonation in neutral isocytosine isomers in the gas phase. They show clearly that the O8H group is the most acidic site in the major neutral isomer iC1a, and the amino groups N3H and N7H possess very close acidities with slight preference of N7H at the DFT level and of N3H at the G4 level in the minor neutral isomer iC3 (Chart 4). For the rare neutral isomer iC2, the amino N1H group exhibits the most acidic properties. However, its ∆acidHi value contributes very little in the macroscopic acidity of the tautomeric mixture.


25


Page 26 of 44

1422.2 (DFT) H 1425.3 (G4) O H

N 1500.5 (DFT) H

N

N

H

1503.6 (DFT) H iC1a {79.6% (DFT) and 97.4% (G4)} 1418.7 (DFT) O 1415.8 (G4) H H N N 1464.2 (DFT) 1417.0 (DFT) 1421.0 (G4) H H N N H N N H H H 1377.1 (DFT) H 1414.5 (DFT) 1428.6 (DFT) 1379.6 (G4) O

H

iC2 iC3 {< 0.1% (DFT) and < 0.1% (G4)} {20.4% (DFT) and 2.6% (G4)}

Chart 4. Microscopic proton acidities in the gas phase (∆acidHi in kJ mol-1 for 298 K) estimated for the OH and NH sites in selected isocytosine isomers. DFT data given in red and G4 data in blue (for selected sites).

The macroscopic acidity (∆acidHm) may give some idea on experimental acidity of isocytosine in the gas phase that can be measured by the equilibrium method.110-112 In such case, intramolecular proton-transfers (prototropy) may be faster processes than intermolecular proton-transfers between isocytosine isomers and reference acids (iC + A-

iCa + HA).

The ∆acidHm, calculated at the DFT (1420.3 kJ mol-1) and G4 (1419.4 kJ mol-1) levels for 298 K according to eq. (9), is not very different from the ∆acidH for the major cytosine isomer determined by Lee and co-workers129 by the bracketing experiments (1431 ± 13 kJ mol-1) and additionally by the extended Cooks method (1435 ± 13 kJ mol-1). It is also close to that (1423 kJ mol-1) evaluated earlier for cytosine by Chen at al.131 on the basis of electron impact spectra and acid dissociation constants measured in dimethylsulfoxide. For the canonical form of cytosine, the N1H group is favorably deprotonated. This site of deprotonation has been proved by various quantum-chemical calculations.129,131 For uracil, the same site (N1H) is favored for deprotonation reaction. However, acidity of uracil in the gas phase is slightly stronger (1393 kJ mol-1)131 than that of cytosine because O8 in U has greater electronegativity than N8 in C.

Deprotonation/Protonation of Charged Radicals. It is well recognized that charged radicals of nucleobases are very reactive species.22-24,74-80,132-137 For example, they can gain or lose one hydrogen atom leading to the corresponding protonated or deprotonated ions.74-77


26

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These type of reactions are very fast and exothermic. They can be observed for charged radicals in the gas phase using various MS spectrometers.74-76,90,111,132,133 Charged radicals can also be deprotonated or protonated to the corresponding neutral radicals.22-24,77-80,132-137 Since free proton does not exist, the proton-transfer reactions for charged radicals depend on acidbase properties of other molecules which can gain or lose a proton. When their acid-base properties in comparison to charged radicals are too weak or too strong, proton-transfers can be unfavorable (endothermic) or favorable (exothermic) processes, respectively. The protontransfer processes can be studied experimentally by various spectroscopic techniques (e.g., ESR, ICR, or MS), as well as they can be analyzed theoretically by quantum-chemical methods.77,78,132-144 In the case of isocytosine charged radicals, the following reactions are possible: iC+• + H•

iCH+, iC-• − H•

iCa, iC+• − H+

diC•, and iC-• + H+

piC•. The favored

isomers for charged radicals (iC+• and iC-•) are given in Scheme 1, and those for protonated (iCH+) and deprotonated (iCa) isomers are shown in Scheme 2. To complete our studies for charged radicals, quantum-chemical calculations have been carried out for selected isocytosine neutral radicals (isomers diC• and piC•, Table S9 in SI), and the favored isomers could be found in the gas phase {DFT(B3LYP)/6-311+G(d,p) and G4 for major isomers) and aqueous solution {PCM(water)//DFT(B3LYP)/6-311+G(d,p)}. For investigations, the favored iC+• and iC-• tautomers have been selected and the sites of deprotonation (endo N3H, exo N7H, or exo O8H) and of protonation (endo N1 or N3, or exo O8), analogous to those for neutral isocytosine, considered. According to DFT results, N7H is the preferentially deprotonated group. Detachment of the exo amino H+, placed at the synperiplanar position to the endo N3 atom in iC3+•, leads to the isomer diC3b• which predominates in the isomeric diC• mixture. For protonation reaction, N3 in iC8-• is the preferred site. Its protonation leads to the major neutral radical piC8b•. The amounts of the major neutral radicals, deprotonated diC3b• and protonated piC8b• isomers are predicted to be equal to 100% in the gas phase (Scheme 3). These amounts seem to be slightly reduced in aqueous solution (to 88.8 and 98.3%, respectively, at the PCM level) in favor of the other radical isomers (4.1 % of diC1aa/3a•, 7.1% of diC3c•, and 1.7% of piC8a•, respectively).


27


(a) Deprotonation of iC+• +. iC1a

+

iC3 +H

O H

H

N H2N

N

H

N

.

H

diC1aa/3a Phase Gas Water

.

+

+

-H O N

N

H H

H

N

H

.

diC3c

%

%

100 (DFT) 100 (G4) 88.8 (PCM)

4.1 (PCM)

H

N

N

.

%

.

O H

diC3b

< 0.1 (DFT)

Page 28 of 44

< 0.1 (DFT) 7.1 (PCM)

(b) Protonation of iC-• iC8+H

+

. +

-H

O N H2N

N H piC8a

Phase Gas Water

.

O H H

H

H

H2N

.

% < 0.1 (DFT) 1.7 (PCM)

H H

N N

H

.

piC8b %

100 (DFT) 100 (G4) 98.3 (PCM)

Scheme 3. Isomeric preferences for deprotonated iC+• (a) and protonated iC-• (b) in the gas phase and aqueous solution.

Proton acidity and basicity parameters in the gas phase could be predicted for charged radicals from the thermochemical quantities calculated for the corresponding charged and neutral radicals using the same procedures as those for neutral isocytosine. The DFTcalculated microscopic acidities for potential sites of deprotonation in iC1a+• and iC3+• and the DFT-calculated microscopic basicities for potential sites of protonation in iC8-• are given in Chart 5. For the favored sites, the G4-calculated microscopic parameters (∆acidHi = 905.8 kJ mol-1 for N7H in iC3+• and PA = 1433.1 kJ mol-1 for N3 in iC8-• ) are also included in this chart. They are not very different from those estimated at the DFT level (905.2 and 1434.0 kJ mol-1, respectively). As it could be expected, acidity and basicity parameters of charged radicals strongly increase in the gas phase in comparison to neutral isocytosine. Analogous increase effects have also been observed for acid-base properties of charged radicals of


28

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nucleobases, their derivatives, and other molecules in the gas phase as well as in solution.2224,77,78,120,132-137

(a) Microscopic Acidities (∆acidHi) 914.6 (DFT) H N 935.7 (DFT) H

N

O

O 920.1 (DFT) H

H +. N

+.

905.2 (DFT) H N N H 905.8 (G4) H 928.8 (DFT)

H

939.0 (DFT) H iC1a +

H

N

.

. iC3+

(b) Microscopic Basicities (PAi) 1403.0 (DFT) O 1380.2 (DFT) H H

1434.0 (DFT) 1433.1 (G4) N - . H N N

H

H 1391.0 (DFT) iC8 -

.

Chart 5. Microscopic proton acidities (a) and basicities (b) in the gas phase (∆acidHi and PAi, respectively, in kJ mol-1 for 298 K) estimated for the OH and NH sites in iC1a+• and iC3+• and for the O and N sites in iC8-•. DFT data given in red and G4 data in blue (for favored sites).

∆acidH/PA [kJ mol-1] Acids

Bases

- 2000 A C H2 O - 1500 I piC iC D I + 1000 T iC+ iCH Y H 3 O+ - 500

.

.

-0

B A S I C I T Y

2000 OH1500 -

iCa iC

1000 -

iC

500 -

.

-

.

diC

H2 O

0-

Figure 6. Comparison of the proton acidities and proton basicities for different forms of isocytosine in the gas phase.

The use of different symbols for proton acidity (∆acidHi) and proton basicity (PAi) in the gas phase is only formal. Both the ∆acidH and PA values can be placed on the same ∆H scale for deprotonation reaction of neutral and ionic species, because ∆acidH(iC) = PA(iCa), ∆acidH(iCH+) = PA(iC), ∆acidH(iC+•) = PA(diCa•), and ∆acidH(piC•) = PA(iC-•). Stronger acids possess smaller ∆acidHs and stronger bases have higher PAs. Comparison of the DFTACS Paragon Plus Environment

29


Page 30 of 44

and G4-calculated enthalpies of deprotonation reactions for different forms of isocytosine isomers indicates that ∆acidH of iC+• is close to ∆acidH of iCH+ and PA of iC-• is close to PA of iCa (Fig. 6). In other words, PA of diC• is close to PA of iC and ∆acidH of piC• is close to ∆acidH of iC.

iC3+• + H2O

iC3+• + 11H2O

iC8-• + H2O

iC8-• + 11H2O

Figure 7. DFT-structures for adducts of major charged isocytosine radicals with one and eleven water molecules.

When proton acid-base quantities of isocytosine charged radicals (Chart 5) are compared to those of water in the gas phase110 (PA = 691 kJ mol-1 and ∆acidH = 1622 kJ mol1

), it is clear that due to very high energy gaps this molecule cannot be spontaneously

protonated by iC+• and also it cannot be spontaneously deprotonated by iC-•. Both intermolecular proton-transfer reactions, iC+• + H2O

diC• + H3O+ and iC-• + H2O

piC• + OH-, are highly endothermic in the gas phase. Water is too weak base and cannot attach a proton from iC+•. Water is also too weak acid and cannot protonate iC-•. The Gibbs energy differences for these reactions calculated for the major isomers iC3+• and iC8-• at the DFT(B3LYP)/6-311+G(d,p) level are equal to 217 and 199 kJ mol-1, respectively. The same is


30

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true in aqueous solution. Both reactions are endothermic at the PCM(water)//DFT(B3LYP)/6311+G(d,p) level (energy gap 122 and 67 kJ mol-1, respectively). However, the major isomers of charged radicals (iC3+• and iC8-•) can form stable H-bonded adducts with both one water molecule {energy gain of 34 kJ mol-1 at the DFT(B3LYP)/6-311+G(d,p) level} as well as with eleven water molecules (Fig. 7), whereas adducts of the neutral deprotonated and protonated radicals (diC3b• and piC8b•) with H3O+ and OH-, respectively, are not found at the two levels of theory. The same is true for adducts of charged radicals with NH3 (PA = 854 kJ mol-1 and ∆acidH = 1683 kJ mol-1).110 Ammonia has not sufficient acid-base strength to deprotonate iC+• and to protonate iC-•. One NH3 molecule (like one H2O) can only form stable H-bonded adducts with iC3+• and iC8-• (Table S12 in SI). Other amphiprotic compounds, possessing acid-base properties similar to those of isocytosine charged radicals, may participate in the proton-transfer reactions with these species. Looking for other bases suitable for the protontransfer reactions, we tested amphiprotic formamidine (NH2−CH=NH, PA(Z-isomer) = 943.1 kJ mol-1 at the G2 level)111 and guanidine {(NH2)2−C=NH, PA = 986.6 kJ mol-1 at the G2 level}.111 Interestingly, the proton-transfer is possible in their H-bonded adducts with iC3+• (see structures in Table S12 in SI). The other adducts, formed by proton-transfer from the exo amino group in iC3+• to the imino N atom in formamidine and guanidine, possess lower Gibbs energies {by 7.4 and 39.1 kJ mol-1, respectively, at the DFT(B3LYP)/6-311+G(d,p) level} than those of neutral bases with iC3+•. Hence, in the equilibrium mixture of the two Hbonded adducts HAB⋅⋅⋅iC3+• (HAB = formamidine or guanidine) and HABH+⋅⋅⋅diC3•, the last one seems to be favored (Fig. 8). Its amount is equal to 95.3 and 100% for the formamidinium and guanidinium adducts, respectively. Unfortunately, formamidine and guanidine have not sufficient acidity {∆acidH = 1526.2, 1537.6, and 1530.0 kJ mol-1 for Z-NH2−CH=NH, ENH2−CH=NH, and (NH2)2−C=NH, respectively, at the DFT(B3LYP)/6-311+G(d,p) level} to protonate the major isocytosine radical anion iC8-• (PA = 1434 kJ mol-1 for N3 at the same DFT level). The proton-transfer is possible at the DFT level in the adduct of formamidine with iC8-• (Table S12 in SI), but the amount of the formamidinium adduct with protonated radical anion piC8• is ca. 1.5·10-4%.


31


HAB⋅⋅⋅iC3+•

HABH+⋅⋅⋅diC3•

iC3+•• + NH=CH-NH2 (4.7%)

diC3• + NH2+=CH-NH2 (95.3%)

iC3+•• + NH=C(NH2)2 (< 0.01%)

diC3• + NH2+=C(NH2)2 (100%)

iC8-•• + NH=CH-NH2 (100%)

Page 32 of 44

piC8• + NH2+=CH-NH2 (< 0.01%)

Figure 8. Proton-transfers in adducts of the isocytosine radical cation iC3+• with formamidine and guanidine observed at the DFT(B3LYP)/6-311+G(d,p) level.

Comparison of Tautomeric Equilibria in Neutral and Ionized Pyrimidine Bases (iC, C, and U). Pyrimidine bases (isocytosine, cytosine, and uracil) display analogous tautomeric equilibria between nine tautomers (1-9 in Scheme 4).41-45 Substituted at 2- and 4positions by −NH2/=NH and/or −OH/=O, they possess two labile protons and five conjugated sites between which the protons can be transferred. Although the schemes of tautomeric conversions are analogous for pyrimidine bases, tautomeric stabilities and consequently tautomeric preferences are not the same for neutral and ionized forms. The main reason is that pyrimidine bases differ by the exo groups. Uracil have two −OH/=O groups, while cytosine and isocytosine, being constitutional isomers, contain one −OH/=O group and one −NH2/=NH group, which possess different acid-base properties.


32

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Y H

H H

N

X

N

H

7 8 YH

YH H

N X

N3

N

HX 7

H

4

HX Y

N

H

X

1

N

N

H

Y H

H

H

HX

H

N N

H YH

3

2

Y H

H

8

H

N

5

H H H

N

H

Y N

HX

YH H

5 2 1 6

H

N

4

X

H H

N H

N

X N

H

N

H

9

H

6

Scheme 4. Tautomeric equilibria between nine tautomers of pyrimidine bases: iC (X: NH, Y: O), C (X: O, Y: NH), and U (X, Y: O).

For illustration of different orders of tautomeric stabilities in neutral and ionized pyrimidine bases, the isomers with the favored conformation of the exo −OH group and/or with the favored configuration of the exo =NH group have been selected for 1-9. Fig. 9 shows variations of their relative Gibbs energies calculated in the gas phase at the same DFT level (Table S3, Figs S5 and S6 in SI). Note that smaller relative Gibbs energy indicates greater tautomeric stability. Generally, the NH-OH and/or NH-NH tautomers (1-6) of pyrimidine bases have smaller relative Gibbs energies (grater stabilities) than the CH ones (7-9) for the positively ionized bases. Situation changes for the negatively ionized molecules, for which the very rare CH form 7 or 8 displays greater stability than most of the NH-OH and NH-NH tautomers. Detailed analysis of the relative Gibbs energies for the three pyrimidine bases (iC, C, and U) shows evidently that the tautomeric preferences are different for both neutral and ionized forms. For neutral bases, they strongly depend on exo groups and their positions in the pyrimidine ring. These groups influence acid-base properties of individual isomers and all possible intramolecular interactions. For charged radicals, their energetic stabilities depend additionally on ionization mechanisms, ionization energies, and electron affinities. All these factors decide about composition of the tautomeric mixture in the gas phase (Table 1).


33


Page 34 of 44

a) Neutral forms ∆G [kJ /mol] for neutral tautomers 1-9 200 Isocytosine

Cytosine

Uracil

5

7

150 100 50 0 1

2

3

4

6

8

9

8

9

8

9

b) Radical cations ∆G [kJ /mol] for radic al c ations 1-9 250 Is oc ytos ine

C ytos ine

Urac il

200 150 100 50 0 1

2

3

4

5

6

7

c) Radical anions ∆G [kJ /mol] for radical anions 1-9 200 Is oc ytos ine

C ytos ine

Urac il

150 100 50 0 1

2

3

4

5

6

7

Figure 9. Change of energetic stabilities (∆G for 298 K) for neutral tautomers 1-9 (a), their radical cations (b) and radical anions (c) of isocytosine, cytosine, and uracil. Table 1. Amounts (in %) of Major and Minor Isomers for Neutral and Ionized Pyrimidine Bases in the Gas Phase Neutral

Radical cation Radical anion Isomer xa Isomer xa +• -• 6.5 0.4 iC1a iC7a 93.5 99.6 iC3+• iC8-• Cytosined,e 23.1 5.5 C1a+• C4-• +• 11.8 0.1 C1b C6b-• 0.1 25.1 C3ab+• C7a-• 16.5 69.2 C4+• C7b-• +• 45.4 C5 0.4 C6a+• 2.6 C6b+• Uracilf,g 100 98.6 2.9 U6 U3a+• U6-• +• 1.4 97.1 U6 U7-• a Calculated at the B3LYP/6-311+G(d,p) level. b For structures see Scheme 1 and Fig. S2. c Ref. 42. d For structures see Fig. S5 in SI. e Ref. 43. f For structures see Fig. S6 in SI. g Refs. 44,45. Base Isocytosineb

Isomer iC1a iC3 C1a C1b C4 C6a C6b

xa 79.6c 20.4c 8.2 2.5 87.0 0.1 2.1


34

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As discussed above, the tautomeric mixtures of neutral and positively ionized isocytosine in the gas phase contain mainly the amino-hydroxy tautomer 1 (isomer a) and the amino-oxo tautomer 3 (Scheme 1). The tautomer 1 is favored for iC, while it is a minor form for iC+•. Situation is reversed for the canonical tautomer 3. It is a favored form for iC+•. The tautomeric mixture of negatively ionized isocytosine consists mainly of the CH tautomers 7 (isomer a) and 8, where 8 exhibits greater stability. When going from isocytosine to cytosine, the exo groups change their positions. This replacement of the functional groups affects the composition of the tautomeric mixture for both the neutral and ionized base. Three tautomers, the amino-hydroxy form 1 (isomers a and b), the amino-oxo form 4, and the imino-oxo form 6 (isomers a and b), predominate for C in the gas phase,43,145 whereas almost all NH-OH and NH-NH isomers, except the hydroxy-imino tautomer 2, are significant for C+•: 1 (isomers a and b), 3 (isomer ab), 4, 5, and 6 (isomers a and b).43 The canonical (amino-oxo) tautomer 4 has the greatest stability for C, and the other amino-oxo tautomer 5 for C+•. The tautomeric mixtures of C-• contain three tautomers: 4, 6 (isomer b), and 7 (isomers a and b), among which the CH tautomer 7 (isomer b) is favored.43 In the case of uracil,44,45 the canonical (dioxo) tautomer 6 is exceptionally stable for U, U+•, and U-•. It exhibits the greatest stability for U and U+•, but for U-• the CH tautomer 7 has lower Gibbs energy than 6. Important stability displays also the hydroxy-oxo tautomer 3 (isomer a) for U+•. All these DFT results show that ionization affects the proton-transfer equilibria in pyrimidine bases in different way and can be responsible for modifications in the DNA or RNA structure.

CONCLUSIONS Quantum-chemical calculations carried out for isomers of ionized isocytosine at three levels of theory, B3LYP/6-311+G(d,p), G4 (for selected isomers), and PCM(water)//B3LYP/6311+G(d,p) for 298 K, gave the possibility to study the tautomeric system from two points of view, microscopic (kinetic) for individual tautomers (isomers) and macroscopic (thermodynamic) for the tautomeric mixture. These investigations showed significant changes


35


Page 36 of 44

of proton-transfer equilibria, and consequently, important changes in the composition of the tautomeric mixture when proceeding from neutral to ionized forms as well as from apolar to polar environment (Scheme 1). The canonical amino-oxo tautomer is only present in the tautomeric mixture of iC and iC+•, while it is a rare form for the iC-• mixture. For negatively ionized base, one of the CH tautomers has the lowest energy and becomes a favored form. The amino-hydroxy tautomer is present in sufficient amounts only in the gas phase for iC and iC+•. In aqueous solution, it is a rare form. Comparison of DFT results for isocytosine with those reported previously for cytosine and uracil containing various groups at 2- and 4-positions43-45 showed additionally important differences in ionization effects on proton-transfers in pyrimidine bases, and consequently on energetic stabilities of the tautomeric forms (Fig. 9). This observation is meaningful for apolar environment (e.g., lipids), where charged radicals may not be protonated or deprotonated to neutral radicals as easily as in aqueous solution,33,74-80 and where they may live longer time. Generally, proton basicity and acidity, estimated in the gas phase in the enthalpy scale for the tautomeric isocytosine mixture (933.7 and 1419.4 kJ mol-1 at the G4 level, respectively) are of the same order of magnitude as those measured for cytosine.129,131 However, the favored site of protonation (N1 in iC and N3 in C) and the favored site of deprotonation (O8H in iC and N1H in C) are completely different in these two pyrimidine bases. Interestingly, when proceeding from the gas phase to aqueous solution, the tautomeric preferences change not only for neutral and protonated isocytosine,42 but also for its deprotonated form (Scheme 2). Ionization increase acid-base properties of charged radicals in comparison to neutral isocytosine. However, their proton acidity and basicity are close to those of the corresponding protonated and deprotonated forms of neutral isocytosine, i.e., acidity of iC+• is close to that of iCH+ and basicity of iC-• is close to that of iCa. Consequently, the H-bonded adducts of iC radicals with amphiprotic compounds of similar acid-base properties confirm the protontransfers possible in the redox nucleic acid base pairs and their methyl derivatives, when one base is ionized by one-electron oxidation or by one-electron reduction.138-144 The labile proton can move from radical cation to neutral base or from neutral base to radical anion, between


36

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the sites of analogous acid-base properties according to the ∆acidH/PA scale, i.e., when the energy gap for proton-transfer in ionized pairs is favorable. For example, taking into account the proton acid-base properties of the canonical neutral and radical forms of isocytosine (guanine building block) and those of 1-methylcytosine studied previously at the same DFT level,121 the labile proton at N3 in iC3+• (∆acidH = 920 kJ mol-1) can move to the N3 atom in neutral 1-methylcytosine (PA = 971 kJ mol-1), and also the labile proton at N3 in iC3 (∆acidH = 1419 kJ mol-1) can move to N3 in radical anion of 1-methylcytosine (PA = 1468 kJ mol-1). Both proton-transfers seem to be exothermic (energetic gain ca. 50 kJ mol-1).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Canonical and rare forms of nucleobases; possible tautomers of guanine connected to sugar; possible isomers of isocytosine and its different forms; isolated (DFT) and solvated (PCM//DFT) structures, atom coordinates, electronic energies, relative thermochemical quantities for isomers of different forms of isocytosine; microscopic ionization potentials, microscopic electron affinities, and microscopic deprotonation enthalpies of isocytosine; adducts of isocytosine radicals with water and other simple bases; HOMO, SOMO and LUMO for selected isocytosine isomers; thermochemical quantities calculated at the G4 level (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: 48 22 5937623. Fax: 48 22 5937635 Notes The authors declare no competing financial interest.


37


Page 38 of 44

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5. 6. 7. 8.

9. 10.

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12.

13.

14.

15. 16. 17. 18.

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Page 44 of 44

Graphical abstract

Acidity and basicity in the gas phase ∆acidH PA Acids Bases

1

.

piC

H2O iC

. iCH

iC+

+

H3O+

- 2000 A C - 1500 I D I - 1000 T Y - 500 -0

B A S I C I T Y

[ kJ/mol ]

2000 OH1500 1000 500 -

. . diC

8

iCa iC

iC H 2O

0-


44

Effects of Positive and Negative Ionization on Prototropy in Pyrimidine

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