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Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

Introduction
DNA is continuously damaged by reactive oxygen species (ROS) generated by UV light, ionizing radiation and during metabolism [1][2][3][4][5]. Among the various products of oxidative stress, 7,8-dihydro-8-oxoguanosine (oxoG) is the most commonly found. It is a pre-mutagenic DNA lesion since oxoG is able to mispair with adenine, thus generating G/C to T/A transversion mutations [6]. As is the case of most oxidized bases, oxoG is primarily removed in the base excision repair (BER) pathway. This pathway is initiated by the recognition of the modified bases by specific DNA glycosylases. In human cells, 8-oxoguanine DNA glycosylase hOGG1 accomplishes the excision of oxoG residues. HOGG1 belongs to the HhH-GPD superfamily of DNA glycosylases containing conserved structural helix-hairpin-helix and GPD motifs [7][8][9][10]. HOGG1 is a bifunctional enzyme, possessing DNA glycosylase activity (hydrolysis of the N-glycosidic bond of the damaged nucleotide resulting in formation of the abasic product, Fig. 1) and AP lyase activity (elimination of the 39phosphate, often referred to as b-elimination resulting in formation of the nicked product, Fig. 1). In contrast to several other bifunctional DNA glycosylases, the AP lyase activity of hOGG1 is much weaker than its glycosylase activity [11][12][13][14].
The 3D structures of free hOGG1 and its covalent complexes with DNA have been determined by X-ray crystallography [9,10,[15][16][17][18][19]. The structural data indicate that the conformations of both enzyme and DNA undergo significant changes upon binding. The enzyme forms non-specific electrostatic and hydrophobic contacts with the sugar-phosphate backbone. In the specific lesion recognition complex the DNA is kinked by 70u. The oxoG base is fully flipped out from the DNA helix and deeply inserted into the active site of the enzyme ( Fig. 2A). Amino acids Arg-154 and Arg-204 inserted in the DNA helix participate in bidentate hydrogen-bonding interactions with the cytosine base, located opposite to the damaged base (Fig. 2B). Additionally, amino acids Asn-149 and Tyr-203 are inserted into the abasic void released due to flipping out of the oxoG residue.
According to the structure of the complex of hOGG1 with a non-specific DNA, the non-damaged G base is held in the semieverted transition state (Fig. 2C) [17]. In this state the base is located in the exo-site at the distance of ca. 5 Å from the catalytic enzyme pocket. Nevertheless, in a recent paper from Verdine's laboratory, a different structure of the complex hOGG1 with non-damaged DNA is proposed (Fig. 2D) [19]. It was shown that although a non-damaged G base can be placed into the active site of hOGG1 approximately in the same position as an oxoG base in the lesion recognition complex ( Fig. 2E and 2F), the catalytic step does not occur with DNAs containing non-damaged G base [16].
Therefore the question arises -what is the mechanism of recognition of the damaged nucleotide?
To address this issue, in the previous studies we investigated the conformational dynamics of hOGG1 and DNA substrates during the catalytic cycles using fluorescence detection [14,20]. The enzyme molecule contains 10 fluorescent tryptophan amino acids, located in several parts of the spatial structure. The fluorescence changes of Trp residues in hOGG1 as well as 2-aminopurine (aPu) residues in the DNA substrates have been detected during catalytic cycle at single-turnover conditions. It was shown that the interaction proceeds through multiple conformational transitions, attributed to sequential lesion recognition process, catalytic steps and release of the product from the DNA complex with enzyme [14,20]. A kinetic scheme (Fig. 3) containing three initial reversible recognition steps, two irreversible chemical steps and final reversible step was proposed to explain the observations. The structural nature of these steps was suggested based on the analysis of several mutant forms of hOGG1 as discussed in detail in [21]. These studies resulted in atomic-level structural and kinetic information to understand the sequence of hOGG1-DNA interactions during the recognition of oxoG base, and provided insights into the roles of individual amino acids in this process. The analysis of the fluorescence kinetic data lead to the conclusion that the first step of the oxoG-DNA repair is a non-specific binding process which consists of C base pulling by Arg-154/Arg-204, pushing of Tyr-203 into DNA helix, electrostatic interaction between Lys-249 and the damaged nucleotide, oxoG flipping out in the exo-site, and the interaction of His-270 with the everted base [21]. During the second step the oxoG base is everted into the active site and is held there due to the p-p stacking with the aromatic ring of Phe-319. After that, His-270 interacts with phosphate group of the everted nucleotide and forms hydrogen bonds between Arg-154, Arg-204, Asn-149 and the C base. The third DNA damage recognition step is most likely associated with the full insertion of Tyr-203 in the duplex and oxoG interaction with Gly-42.
Understanding of the mechanism of the DNA damaged base recognition by hOGG1 may be improved by elucidation of the thermodynamics of the binding and cleavage steps via determination of their Gibbs free energy (DGu), enthalpy (DHu) and entropy (DSu). Correlation of the thermodynamic data with the proposed sequential mechanism of oxoG-substrate binding and cleavage might result in the quantitative description of the driving forces that govern the structural adjustment of the enzyme and DNA. In the present work, the thermodynamic parameters of the specific steps occurring in the time range 1 ms-1000 s during oxoG base recognition and removal by hOGG1 were obtained via the temperature-dependent fluorescence kinetic data using the van't Hoff and Eyring equations [22]. The rate constants of the   [17]), (B) specific contacts of enzyme with the cytosine which is located opposite to oxoG base (PDB ID: 1EBM, [9]), (C) the complex of hOGG1 bound to the DNA with the G base inserted in the exo-site (PDB ID: 1YQK, [17]), (D) the complex of hOGG1 bound to the DNA, with the G base inserted in the active site (PDB ID: 3IH7, [19]). The overlays of the trapped catalytic complex hOGG1/oxoG-substrate (red, 1HU0, [16]) with either (E) the complex of hOGG1 bound to DNA with the G base inserted in the exo-site (blue, 1YQK, [17]) or (F) the complex of hOGG1 bound to DNA, with the G base inserted in the active site (green, 3IH7, [19] forward and reverse reactions as well as the resulting equilibrium constants of the individual steps in a multi-step reaction mechanism were determined based on the temporal 2-aminopurine (aPu) fluorescence traces recorded at different temperatures. This fluorescent analog of purine bases is sensitive to the DNA conformation and was widely used for the studies of structural changes in DNA (see [23,24] for details).

Protein Expression and Purification
To purify hOGG1 expressed as a recombinant protein, 1 liter of Escherichia coli strain Rosetta II(DE3) (Invitrogen, France) carrying the pET28c(+) hOGG1 construct was grown in LB broth with 25 mg/ml kanamycin at 25uC until A600 = 0.6-0.7 and induced overnight with 0.2 mM isopropyl-b-D-thiogalacto-pyranoside. The method of the isolation of the wild-type hOGG1 was described previously [14,21]. The protein concentration was measured according to the Bradford method [25]; the stock solution was stored at 220uC.

Oligodeoxynucleotides
The sequences of ODNs used in this work are listed in Table 1. The ODNs were synthesized by standard phosphoramidite methods using an ASM-700 synthesizer (BIOSSET Ltd., Novosibirsk, Russia) in the Laboratory of Bionanotechnology of ICBFM from phosphoramidites purchased from Glen Research (Sterling, VA). Synthetic oligonucleotides were unloaded from the solid support with ammonium hydroxide according to the manufacturer protocols. Deprotected oligonucleotides were purified by HPLC. The purity of ODNs exceeded 98% as estimated by electrophoresis in 20% denaturing PAGE after staining with the Stains-All dye (Sigma-Aldrich). Concentrations of oligonucleotides were determined from their absorbances at 260 nm. The ODN duplexes were prepared by annealing of modified and complementary strands at the 1:1 molar ratio in the reaction buffer (50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 9% glycerol).

Stopped-flow Fluorescence Measurements
Stopped-flow measurements with fluorescence detection were carried out essentially as described previously [14,20,21]

Product Analysis
To analyze the products formed by hOGG1, the oxoGsubstrate was 59-32 P-labeled using T4 polynucleotide kinase and [c-32 P] ATP. The reaction mixtures contained reaction buffer, 1.0 mM 32 P-labeled oxoG-substrate and 2.0 mM enzyme. The reaction was initiated by adding the enzyme and allowed to proceed at different temperatures (10-30uC). Aliquots (2 ml) were withdrawn as required, mixed with 3 ml of gel-loading dye containing 7 M urea, and analyzed by 20% denaturing PAGE. The gels were exposed to Agfa CP-BU X-ray film (Agfa-Geavert), and the autoradiograms were scanned and quantified using Gel-Pro Analyzer, version 4.0.

Data Processing
The processing of the fluorescence intensity traces was performed by numerical fitting of a solution of a system of ordinary differential equations corresponding to the reaction mechanism using DynaFit software (BioKin, Pullman, WA) [26] as described previously in [27][28][29][30].
The approach is based on the fluorescence intensity variation in the course of the reaction due to the sequential formation and further transformation of DNA-enzyme complex and its conformers. The software performs numerical integration of a system of ODE with subsequent non-linear least-squares regression analysis. The response factors of the intermediates (that are essentially the products of the molar extinction coefficients and the fluorescence quantum yields) are treated as fitting parameters in the data processing. In the evaluated mechanisms, except for the first bimolecular step, all other reactions are first order reactions. In the data processing, the kinetic information is obtained from the . Kinetic mechanism of hOGG1 processing of the oxoG-substrate. Here E is hOGG1; OG is the oxoG-substrate; (E OG) n are different enzyme-substrate recognition complexes; E AP is the complex of E with the abasic site formed in the course of N-glycosylase reaction; E P is the enzyme-product complex formed in the AP-lyase reaction; P is the final free reaction product; k i and k -i are the rate constants of the forward and backward individual processes. doi:10.1371/journal.pone.0098495.g003 'temporal behavior' of the fluorescence intensity, not from the 'amplitudes' of the specific signal contributions. Only the rate constants obtained in the non-linear fits were used to derive the thermochemical parameters. The 'response factors' for different conformers resulting from the fits were not used in the determination of the equilibrium constants, but rather provided additional information on the fluorescence intensity variations in different complexes and conformers. Processing of individual kinetic curves does not unambiguously provide the kinetic parameters; therefore, global fits of sets of kinetic curves obtained at different concentrations of the reactants at each temperature was used. In the fits all relevant rate constants for the forward and reverse reactions, as well as the specific molar responses for all intermediate complexes were optimized.
Using the measured rate constants the equilibrium constants K i (k i /k -i , i is the step number) were determined for G-and oxoGsubstrate. The standard thermodynamic functions of the i-th equilibrium step were determined using the van't Hoff equation, which represents the relation between the true thermodynamic equilibrium constant (K i ) and the Standard Gibbs Energy, the Standard Enthalpy, and the Standard Entropy of the i-th reaction step [22]:  Table 3. Thermodynamic parameters of hOGG1 interactions with oxoG-substrate a .
Step The typical dependence ln(K i ) versus 1/T was linear, as expected for the relatively narrow temperature range of the study. The Gibbs free energies DGu i at 25uC were calculated from ln(K i ) = 2DGu i /RT. The validity of this approach was discussed elsewhere (e.g., Ref. [31]).
Analysis of the temperature dependence of the rate constant of a chemical reaction k i permits determination of the standard enthalpy of activation (DHu ,{ ) and the standard entropy of activation (DSu ,{ ) based on the Transition State Theory (Eyring equation [22]). For unimolecular reactions, such as the catalytic step 5 in the reaction mechanism, where k B and h are Boltzmann and Planck's constants, respectively, R is the gas constant, T is absolute temperature in Kelvins, k i is the rate constant of the chemical step i.

Molecular Modeling
The X-ray data obtained for complexes of hOGG1 with G and oxoG containing DNA ( [17], PDB ID 1YQK and 1YQR, respectively), are taken as models of atomic structures of protein-DNA complexes (ENOG) 1 and (ENOG) 3 in Figure 3.
Inspection of these structures revealed, that structure 1YQK represents an example of the initial protein-DNA complex (ENOG) 1 , with bended DNA interacting with the protein and the G base placed in the exo-site. Structure 1YQR represents the specific protein-DNA complex (ENOG) 3 with oxoG base everted from the double helix into the enzyme's active site, with filled void in the double helix by Arg-154, Arg-204, Tyr-203 and Asn-149 and the enzyme forming a tight complex with the DNA-substrate in the catalytically active conformation. The contact areas DMS of the protein-DNA interface were calculated as the difference of the molecular surface (MS) confining the solvent-excluded volume [32] of isolated protein and the DNA and the protein-DNA   [33]. The free energy of the cavity formation in water in the process of desolvation has entropic nature and it is approximated by the linear equation DG cav = cDMS, where the parameter c is in the range of 70-117 cal/mol/Å 2 [32]. For the evaluation of the entropy loss, c = 80 cal/mol/Å 2 was accepted (which approximates the entropy of desolvation of non-polar species in water [34]).

Interactions of hOGG1 with oxoG-substrate
The thermodynamics of the oxoG recognition by hOGG1 provided very important information for identification of the key steps in the DNA lesion search process. To quantitatively characterize the thermodynamics of the damage recognition process the changes in the fluorescence of an aPu label on 59side from oxoG base were used to reveal the conformational changes of DNA in the close proximity to the damaged base. The fluorescence traces measured for various concentrations of enzyme at each temperature ( Fig. 4A and 4B) were fitted by the kinetic mechanism shown in Figure 3, as in the previous studies [14,20,21]. The rate constants obtained from the fits are summarized in Table 2. The thermodynamic parameters (Table 3) were then calculated using the van't Hoff analysis (Fig. 5) as described in Materials and Methods.
The effect of temperature on the rate of the chemical reactions was determined in the direct measurements of the rate of the products formation (abasic product in N-glycosylase reaction and DNA chain nick in AP-lyase reaction) using PAGE analysis of 32

Interactions of hOGG1 with Non-specific DNA
The aPu fluorescence increases substantially during the process of hOGG1 binding of the non-specific G-ligand; the process is essentially completed in about 0.2 s (Fig. 6). The observed increase in the fluorescence intensity of aPu indicates local melting of the DNA chain interior of the non-specific enzyme-DNA complex. As it is known from the structural data obtained for covalently linked complex hOGG1 with a non-specific DNA, the enzyme is capable to flip out the undamaged G base not only in the exo-site [17] but also in the active site [19]. According to the kinetic data obtained for a set of hOGG1 mutant forms [21], the non-specific binding includes pulling of the opposite C base with Arg-154 and Arg-204, pushing by the Tyr-203 residue into the duplex between cytosine and the next base, the H-bond formation between the His-270 residue and the phosphate group of the damaged nucleotide, and stacking of the G base with the Phe-319 residue. All these factors lead to disruption of the stacking between aPu and G bases due to the flipping out of the latter and the DNA bending, which are expected to enhance aPu fluorescence. The aPu fluorescence traces obtained for G-ligand do not exhibit decreases in the fluorescence intensity as would be indicative of the void-filling processes. This means that the full insertion of the G base in the active site of hOGG1 as well as interaction of the opposite C with Arg-154, Arg-204, Asn-149 and Tyr-203 and its insertion into the DNA void, as suggested by the structural studies [19], is kinetically unfeasible.
The measured fluorescence traces at various concentrations of enzyme at each temperature (Fig. 6) were fitted by a kinetic mechanism containing two binding steps (Fig. 7). The forward and the reverse rate constants as well as the dissociation constant obtained at different temperatures are listed in Table 4. The  Table 5.

Discussion
Comparative analysis of the thermodynamic data presented in Tables 3 and 5 together with the structural information for free hOGG1 and various hOGG1/DNA complexes [9,10,[15][16][17][18][19] provides important information on the energy consumption in the stages of the DNA binding, specific sites recognition and catalysis.
The process of G-ligand binding proceeds in two steps, which could belong to the formation of non-specific electrostatic and hydrophobic contacts with the sugar-phosphate backbone, DNA bending, Arg-154, Arg-204, Asn-149 and Tyr-203 amino acids insertion into the DNA and the G base flipping out into the exo-or active site of the hOGG1, as follows from the X-ray studies [17,19].
The formation of the 1 st complex is favorable in terms of the Gibbs energy change, due to both negative enthalpy and positive entropy changes ( Table 5). The second detectable binding step for G-ligand is characterized by the increase of aPu fluorescence and completed within ca. 0.2 s. As follows from Figure 6, no decrease in the aPu fluorescence was detected, indicating that the full insertion of mentioned above amino acids into the DNA void does not occur. The formation of a tighter 2 nd enzyme-ligand complex is characterized by the decrease of entropy compensated by a favorable change of enthalpy, indicating formation of new contacts, which stabilize this complex and make it more rigid (Table 5). Overall, binding of hOGG1 with G-ligand is thermodynamically favorable process with DGu = 26.9 kcal/mol. Therefore, hOGG1 binding to G-ligand as monitored by direct fluorescence comprises melting of the duplex and eversion of the G base, with the G base left mainly outside the active center. This follows from the absence of kinetic evidence of full insertion of the enzyme amino acids into the DNA duplex, which is expected in the case when the G base is placed in the active site similar to that as for the oxoG base. Therefore, it is reasonable to conclude that in the case of true equilibrium and kinetically restricted state for the hOGG1/G-DNA complex the location of G base in the spatial structure could be different.
The fluorescence traces obtained for oxoG-substrate ( Fig. 4A  and 4B) show that at 0.2 s the aPu fluorescence starts to decrease, which characterizes the insertion of the amino acids. It means that the insertion of amino acids occurs immediately after the eversion of oxoG base from DNA-helix. Therefore, the second step is very important to discriminate G bases vs. oxoG bases. According to [21], specific interactions between Arg-154, Arg-204, Asn-149 and DNA chain occur during this step. Additionally, the oxoG is placed in the active site where it forms stacking structure with Phe-319.
For oxoG-substrate, the formation of the second (ENOG) 2 and the third (ENOG) 3 complexes in Figure 3 is characterized by the positive enthalpy and entropy changes (Table 3). A several processes during the protein/DNA complexation can lead to a significant unfavorable contribution to the enthalpy. The desolvation of polar groups at protein/DNA interface is expected to be energetically unfavorable [35]. Moreover, the protein bindings in the minor groove exhibit positive enthalpy and positive entropy  Table 5. Thermodynamics parameters of hOGG1 interactions with G-ligand a .
Step changes that compensates the process endothermicity. Therefore, these processes are completely entropy driven [36]. The unfavorable enthalpy changes for the minor groove binding proteins result from the displacement of water molecules that are in the minor groove. Because these water molecules are highly ordered, their release into the bulk solution leads to a significant entropy growth.
In the case of hOGG1, the aryl ring of Tyr-203 is wedged into the DNA duplex from the side of the minor groove [9]. Therefore these results are in accord with the conclusion that the enzyme interacts with the minor groove of oxoG-substrate [9]. Another potential source of unfavorable enthalpy DHu is the stress of the DNA structure induced by the ligand attachment. The comparison of the enthalpy and entropy changes for a wide range of DNA-binding proteins [35] shows that complexes in which the DNA is bent or severely distorted exhibit an unfavorable enthalpy changes compensated by favorable entropy changes. According to the structural data, in the catalytically active complex hOGG1 bends the DNA (ca. 70u) at the lesion site. Therefore, DNA bending can be also associated with these steps of oxoG-substrate binding by hOGG1.
The X-ray structures of the complexes of hOGG1 with G (1YQK) and oxoG (1YQR) containing DNA [17] might be considered as the atomic models for the hOGG1-DNA complexes corresponding to the initial complex (ENOG) 1 and the catalytically competent complex (ENOG) 3 , respectively. Therefore, it was possible to estimate the change of the area of the protein-DNA interface during the formation of (ENOG) 3 from (ENOG) 1 . The calculations show that the mutual adjustment of the enzyme and DNA in the complex (ENOG) 3 is accompanied by the increase of the contact surface interface by 672 Å 2 from 764 to 1436 Å 2 in complexes (ENOG) 1 and (ENOG) 3 , respectively. Burring of 672 Å 2 of the molecular surface of the tight protein-DNA complex results in the entropy increase of about 180 cal mol 21 K 21 [34]. This entropy increase is partially compensated by the decrease of entropy due to the formation of the new tight protein-DNA contacts in the course of oxoG-substrate binding steps ( Table 3).
The transition state for the catalytic step of the base excision (Nglycosylase reaction, step 4) as well as the 39-phosphodiester bond cleavage (b-elimination reaction, step 5) has unfavorable enthalpy DHu ,{ and favorable entropy DSu ,{ changes ( Table 3). The results of the thermodynamic analysis of the catalytic steps obtained using aPu fluorescence data were qualitatively confirmed by the direct PAGE analysis of the products accumulation at different temperatures.
As it was recently demonstrated for another oxoG excision DNA glycosylase Fpg from E. coli, belonging to the Fpg/Nei family of DNA glycosylases, the determination of the thermodynamic parameters of the substrate binding and lesion removal steps provided important information about oxoG recognition process [27,29,30]. The pre-steady-state kinetic data were obtained using pyrrolocytosine fluorescence in DNA [27]. These data allowed determination of the Gibbs energy, enthalpy and entropy for the five rapidly proceeding steps of sequential damage recognition mechanism and to develop a detailed model of the Fpg enzymatic process. The first step had similar thermodynamic parameters both for undamaged G-ligand and oxoG-specific substrate, as it was obtained for hOGG1 catalysis in the present study. This step is highly exothermic. In this step the favorable enthalpy change is accompanied by an increase in entropy most probably due to the DNA melting in the point of contact and the conformational changes of the DNA binding site in the Fpg molecule with the emphasis on the contribution of the Phe-110 wedge movement. The next 2 nd , 3 rd and 4 th steps are characterized by the negative or slightly positive DGu changes and reflect the formation of a kink in the DNA chain; eversion of oxoG base from the double helix into the enzyme's active site; and filling the resulting void in the double helix by Arg-108 and Met-73. The 5 th reversible recognition step was characterized by a highly unfavorable enthalpy that was compensated by favorable entropic contribution. The large positive entropy of the protein binding originated from the dehydration of the DNA grooves and isomerization of the enzyme to form a tight complex with the DNA-substrate and to produce catalytically active conformation.

Conclusions
Summarizing, in the present study a thermodynamic analysis of the reaction of human 8-oxoguanine DNA glycosylase with DNA duplexes containing undamaged and damaged nucleotides was performed. It was shown that the first binding step only weakly depends on the nature of the damaged nucleotide, supporting the hypothesis that the main contribution to the thermodynamics of this step is due to the non-specific interactions between the enzyme and the DNA backbone. The results of this study clearly demonstrate that the G base does not evert into the active site, however, do not exclude a possibility for the evertion of the G base in the exo-site suggested earlier by Verdine et al. [17]. The second step is crucial for the discrimination between oxoG and G bases. In the case of oxoG-substrate the second and the third steps have unfavorable enthalpy, which is compensated by favorable desolvation entropy. During these steps displacement of water molecules from the minor groove as well as DNA bending occurs. The last binding step also includes the final adjustment of the enzyme/ substrate complex to the catalytically competent state. This step might be attributed to the full insertion of Tyr-203 in the DNA helix accompanied with the formation contacts between oxoG and Gly-42.
The thermodynamic analyses of the catalytic processes performed by functionally homologous but structurally different DNA-glycosylases, human OGG1 (this work) and E. coli Fpg [27], belonging to two distinct structural families, HhH-GPD and Fpg/ Nei, respectively, demonstrate that they employ the common energetic features in the main steps of 8-oxoguanine lesion recognition. The first step of the DNA binding process is the formation of a non-specific complex, resulting in the double helix melting and insertion of wedging aminoacids (Tyr-203 for hOGG1 and Phe-110 for Fpg), characterized by favorable changes of both enthalpy and entropy. The following steps of lesion recognition are favorable in enthalpy but may be unfavorable in entropy due to the formation of structurally rigid complexes. The last step of the lesion recognition where enzyme-substrate complexes are rearranged to catalytically competent conformations is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of DNA grooves.


Catalytic effect of Fe, Ni, Co and Mo on the CNTs production

In this work a chemical vapour deposition (CVD) process was implemented and automatized for the synthesis of carbon nanotubes (CNTs). Looking the possibility to make an extensive use of biogas, and also for decreasing the costs of the process, methane was used as carbon source. Different CNT structures were obtained using Fe, Ni, Co and also Mo nanoparticles, supported on silica-gel as catalyst for CNT's growing. The number of walls, the morphological effects and efficiency of the process showed dependency on the catalyst type. Cobalt helps the growing of CNTs with fewer layers, compared with Ni, Fe and Mo. Low quality structures are mainly obtained with Fe nanoparticles. Cobalt promotes small diameters in carbon nanotubes and Ni promotes the best quality and yielding during the CVD process.

1.
Introduction Facing the market of nano-materials, one of the main kinds which are increasingly used for different technologies are the CNTs. Due to excellent structural, physical, electrical and mechanical properties, CNTs have stimulated the development of new technologies unimaginable some decades ago. This has become the subject of studies focused on exploring the use of CNTs in various areas [1]. The CNT applications can enable nano-scale molecular levels, which have also generated great enthusiasm in the scientific community and a revolution in modern materials science [2]. Currently is possible to find a variety of studies about CNTs, ranging from structural and physicochemical properties, synthesis to surface modifications [3,4]. Despite this vast literature, the knowledge obtained on the CNTs are just the prelude of its potential.
Historically, CNTs have been obtained by different methods; including: chemical vapor deposition (CVD), solvothermal (several synthetic strategies have been employed without using transition metal catalyst. Templates, precursors and hydro or solvothermal methods, using binary and complex metal 1 *To whom any correspondence should be addressed oxides, chalcogenides, and carbides can be implemented), electrochemical (deposition from organic solutions at room temperature, with formation and growth stimulated by transition metal catalyst, such as iron and/or nickel), or hydrothermal methods (hydrothermal synthesis using polyethylene/water mixtures in the presence of nickel at 700-800° C under 60-100 MPa pressure), as well as arc discharge (soot formed from graphite electrodes during arc discharge), reactive milling (ball-milling and annealing or a pressure-vapor-condenser), laser ablation (a pulsed-laser vaporizes a graphite target in a high-temperature reactor as an inert gas is blend into the chamber), plasma forming (plasma growth at low temperatures) and HiPCO (continuous flow high-pressure carbon monoxide) [5][6][7].
Although all the methods for nanotube synthesis and processing are still under continuous development, the most common synthesis method is the hydrocarbon pyrolysis process, also known as carbon vapor deposition (CVD). For CNTs production, it is usually known as thermal CVD or catalytic CVD, to distinguish it from many other kinds of CVD used for various purposes [8]. CVD is an economically and industrially viable process due to the facility to scale. In this process, CNTs can grow from catalyst particles using batch or continuous processes such as a fluidized bed. Thermal decomposition of a hydrocarbon vapor is achieved in the presence of the metal catalyst. As a carbon source it is possible to use different hydrocarbons compounds, including biogas, allowing a proper kinetic for the production of CNTs [9] and the modulation of the nanotube diameter [10][11].
The catalysts used for the production of CNTs are usually transition metals supported on silica [12], mesoporous silica (MCM-41, MCM 48, SBA-15), [13], calcium carbonate, zeolites [14] or magnesium oxide [15]. All types of catalysts have been studied with the aim of increasing the productivity of CNTs, for improving their properties and modulate their structures. In this work a CVD process was used for the synthesis of CNTs, using different silica-supported catalysts, like Fe, Ni, Co and Mo. The sol-gel method was used for the synthesis of supported metal catalysts.

Catalyst preparation
The sol-gel method has been extensively used in the preparation of supported metal catalysts because it typically results in highly homogeneous materials with high degree of metal dispersion [16]. In this sense, catalysts were supported on silica sol-gel with the metal to 50 percent based on silica added. To obtain metallic catalyst supported on high-surface area silica by the sol-gel method, the polymerization of an alkoxy-silane such as tetrathoxysilane (TEOS), also known as tetraethyl orthosilicate, is carried out in the presence of the appropriate metal precursors. In our case, catalyst nanoparticles were prepared from high purity salts of the  [17]. To accelerate the polymerization, an increase in pH can be brought about by addition of a base, which causes a rapid hydrolysis followed by polymerization [18]. Simultaneously with this polymerization process, the metallic ions (Fe, Ni, Co and Mo) precipitate, thus forming a homogeneous and well-dispersed mixture.

CNTs synthesis
After placing the catalyst inside a quartz tube, a continuous nitrogen flow rate of 1 L/min was passed through the reactor for removing the oxygen. Subsequently, the reduction process was accomplished within at 600º C. The reduction process was kept for 30 minutes in an atmosphere of 90 % v/v of N 2 and 10 % v/v of H 2 . Next, the temperature was increased up to 700º C for the nucleation and growing of CNTs.

Characterization
The high-resolution images were obtained using a high-resolution transmission electron microscope JEOL JEM-2010, operated at 200 kV and a scanning electron microscopy (SEM) JEOL JSM 5300 operated at 5 kV. Complementary RAMAN spectroscopy was performed, and also thermogravimetric analyses (TGA) for CNTs were carried out in a SW Stare Mettler 8.0 device, with a heating rate of 10 °C/min (25 to 900 °C) in an air atmosphere with flow of 50 ml/min.

3.
Results and discussion Raman spectra of CNT samples using different catalysts are presented in Figure 1. Studies carried out by Dresselhaus about Raman spectra for CNT show that the number of defects in graphene layers depends on the ratio I D /I G , which must be less than 0.45 for high structure quality of the CNTs [19]. Figure 1a shows the Raman spectra for CNTs-Ni, in which the ratio I D /I G is 0.416, relating a high purity material. The highest purity was obtained in CNTs-Co, with an I D /I G ratio of 0.26. On the other hand, with Fe, and Mo the quality is decreased in a high level (Figures 1c, 1d), mainly with Fe (I D /I G ~ 1.48). This is due to the low solubility of C in Fe. In order to obtain a better quality, in this case the CVD process must performed to high temperatures (~900°C). In our experiments, for comparison purposes, the temperature was always the same for the different metal-catalyst (~700°C).
According to previous reports [20], the increase of the D band intensity (characteristic peak at ~1350 cm -1 ) with decreasing multiwalled carbon nanotubes (MWCNT) content, is a direct result of the addition of carbonaceous by-products. In the same sense, a decrease in the G' band intensity (characteristic peak at ~2700 cm -1 ) is observed as the MWCNT mass fraction decreases. The G' band on Figures 1a and 1b Figure 2 shows a linear growth of CNTs using Ni as catalyst. The Figure 2a shows that the external diameter distribution of CNTs-Ni ranges from 40 nm to 100 nm. The lengths reached up to several microns (1-7 μm). The tips of these CNTs are closed and contain the catalytic particles enclosed. Figures 2b and 2c show the layers for an isolated CNT. An analysis of the image obtained by HRTEM shown in Figure 2b, lead to conclude that a total of 69 layers and an external diameter of 85.05 nm was obtained (between the layers a gap of ~0,35nm can be calculated in order to obtain the total of layers, according to the methodology developed by Gommes [21] and collaborators, and performing some numerical treatments, through the cross section of the CNTs using the Lambert's law [22]). Also, it is observed that the CNT´s layers are relatively aligned, verifying the structural quality predicted by the I D /I G ratio in Raman spectra (Figure 1).  Figures 3a-3c show CNT-Co. In this case, the number of walls in the samples is less than the rest of catalyst (Fe, Ni, Mo), with a length above 10 μm. Figures 3b and 3c show that the CNTs layers ranged from 4 to 12. Despite the little number of walls, during the CVD process we obtained MWCNT because the number of walls is in average higher than 4 layers. The CNT-Co allows low number of layers as has been reported by S. Porro [23]. Some CNTs have internal diameters less than 10nm and an external diameter less than 50 nm.

a) b) c)
The   Figure 4 shows CNT-Fe with lengths about 4 μm with not high crystallinity, an internal and external diameters of ~ 12 nm and more than ~ 40 nm respectively and 57 layers. The growth of CNT-Fe is tangled and disorderly. According to the results obtained by Huang [24], CNT-Fe and CNT-Co present a non-linear growth. It is also common find the catalyst encapsulated inside the CNT, as a result of the growing process and the little chemical interaction between the catalyst and the silica-gel support. In this sense, the equilibrium state of iron oxide phases (α, β) promote the agglomeration of the catalyst nanoparticles and decrease the encapsulation and the introduction of nanoparticles inside the silica support [25].  Figure 5 shows the CNT-Mo. The micrographs exhibit the low productivity compared with Ni, Co, and Fe. Besides the matrix of silica gel may cover the metal particles, exposing a little surface area to the carbon precursor gas. The main reason for this scarce production of CNTs is attributed to the little solid-state diffusion of carbon in molybdenum. It was difficult to obtain a high purity in samples, dude to the big size of silica clusters (red circle in Figure 5a). The presence of silica during HRTEM analyses inhibits the observation of the numbers of layers due to the electron´s saturation at high voltages (~ 200 kV). Nevertheless, Figure 6 shows the external diameter of CNT-Mo but obtained by FESEM at low voltages. The external diameter ranges between 64.8 to more than 70 nm.

Conclusion
When the sol-gel method is used for the synthesis of supported metal catalysts, additional parameters related to the chemistry of the metal precursors are also important. In this regard, this work showed that the different metals used as catalysts allowed the growing of carbon nanotubes. The CNT-Ni is more homogeneous and stable than CNTs grown with other catalysts. Cobalt allows modular small diameter sizes (7-40 nm) during the sol-gel synthesis. The CNT-Fe was not very stable. In other words, because the diffusion of carbon in iron is favored with increasing temperature (>700° C), the presence of amorphous carbon is promoted. The Mo catalyst requires temperatures above 800° C for the deposition of CNTs. The silica sol-gel is a catalytic support that allows high surface area for different catalytic metals (i.e. Fe, Ni, Co, Mo) facilitating the synthesis of CNTs at low temperatures, with different morphological characteristics, for different potential applications.


s2fieldsofstudy: Chemistry Physics

text:
Molecular tunnelling ionization of allyl halides induced by phase-controlled two-colour laser fields

We have investigated the molecular tunnelling ionization (TI) of four allyl halide molecules (C3H5X; X = F, Cl, Br, I) induced by phase-controlled two-colour laser fields consisting of a fundamental light and a second-harmonic light with a pulse duration of 130 fs and an intensity of 1012–1013 W cm−2. The geometric structure of the highest occupied molecular orbital (HOMO) of the four allyl halides can be systematically changed by replacing the halogen atom. We observed phase-dependent orientation-selective molecular ionization in the four methyl halide molecules. The mechanism of molecular TI is discussed in connection with the geometric structure of the HOMO.

Introduction
Tunnelling ionization (TI) of atoms and molecules induced by intense laser fields occurs when the binding potential of an electron is transformed and suppressed by the electric field of the laser so strongly that the wavefunction of the outermost electron penetrates the potential barrier and the electron is removed from the atom or molecule through the tunnelling process [1][2][3][4][5][6][7]. Recent studies have revealed that TI induced by laser fields with an intensity of more than around 10 13 W cm −2 occurs mainly in the attosecond (1 as = 10 −18 s) time region, when the electric field of the laser reaches its maximum value because of a highly nonlinear optical response [8,9]. TI is one of the most important and fundamental phenomena for observing and controlling physics in the attosecond time regime [10][11][12].
The investigation of molecular TI has made rapid progress both theoretically and experimentally since the introduction of molecular manipulation by intense laser fields. In molecular systems, the concept of the molecular orbital is a basic idea Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. in quantum chemistry [13]. In the molecular orbital picture, the outermost orbital, called the highest occupied molecular orbital (HOMO), plays an important role in understanding not only the diversity of chemical bonds and resultant molecular structure but also the properties of chemical reactions [13]. Recent studies have found that the HOMO is also a major contributor to molecular TI. Ammosov-Delone-Krainov (ADK) theory [3], which is often applied to atoms to compare the absolute values and electric field dependence of ionization rates between theory and experiment, has been extended to treat molecular systems (the molecular ADK model) [14,15]. A qualitative consequence of the molecular ADK model is that the TI rate reflects the geometric structure of the HOMO [16][17][18][19][20]. We can make the physical interpretation that photoelectrons are much more likely to be extracted via tunnelling from the large-amplitude lobe of the HOMO in the opposite direction of the electric field vector. As a consequence of the angular dependence of the TI rate between the electric field vector and the molecular axis, molecules aligned in a certain direction are selectively ionized in a randomly oriented gas-phase molecular ensemble, and the photofragment-emission pattern induced by molecular TI reflects the asymptotic structure of the HOMO [16,17]. As examples of homonuclear diatomic molecules, O 2 and N 2 have, respectively, a butterfly-shaped pattern reflecting the structure of the π orbital and a dumbbell-shaped pattern reflecting the σ orbital structure, as indicated by two-dimensional photofragment-emission pattern imaging [16].
Recently it has been pointed out that another contribution plays an important role in molecular TI: the Stark shift of the ionization potential. The Stark shift reduces or raises the ionization potential when the laser field vector is parallel or antiparallel, respectively, to the dipole of the HOMO, which leads to a maximum ionization rate for the antiparallel configuration where more electrons are ionized from the smaller-amplitude side of the HOMO [21][22][23]. Thus, the effect of the Stark shift trends opposite to that of the geometric structure of the HOMO [21][22][23] (an induced dipole synchronized with oscillating laser fields are always parallel with electric field vector so that the induced dipole has no influence for the orientation-dependent ionization). A theory for molecular TI that considers the Stark effect, called the weak-field asymptotic theory (WFAT) based on the Siegert states in a static electric field, has been developed within the single-active-electron approximation [24,25]. It has been shown that the dominance of either the Stark shift or the shape of the HOMO depends on the molecule and cannot be determined by just looking at the shape of the HOMOs [24,25].
The dominance of either the Stark shift or the geometric structure of the HOMO in molecular TI can decisively influence experimental molecules with asymmetric structure, where the ionization rate depends on the head-tail order of the molecule. Recent experiments with carbon monoxide (CO) on TI induced by intense linearly polarized (LP) two-colour phase-controlled laser fields consisting of a fundamental light and its second-harmonic light ('phase-controlled' means that the relative phase difference is stable and can be externally controlled but not stabilized actively such as phase-stabilized few-cycle pulses. hereafter, LP ω + 2ω laser fields) [26,27] and cold target recoil ion momentum spectroscopy (COLTRIMS) [28] have demonstrated that more tunnelling ionization is observed from the C-side, which has the largeamplitude lobe of the HOMO, than the O-side and that the Stark effect plays a minor role.
On the other hand, the angular dependence of the TI rate for carbonyl sulfide (OCS) molecules exposed to singlefrequency circularly polarized laser fields shows that the ionization rate is maximum when the laser field points from nuclei O to S, indicating that more TI occurs from the O-side, where the HOMO has a smaller amplitude, and that the Stark effect makes a major contribution [21]. Furthermore, it was found that molecular TI in OCS induced by LP ω + 2ω laser fields is dominated by the geometric structure of the HOMO, which is different from the results obtained with singlefrequency circularly polarized laser fields [29]. Although molecular TI is an important and fundamental phenomenon in the field of molecular science using intense laser fields, molecular TI is still controversial in that the mechanisms extracted from some experiments have not yet converged. Considering this situation, it is important to explore various molecules and accumulate experimental results regarding molecular TI.
We have experimentally investigated orientation-selective molecular TI (OSM-TI) induced by LP ω + 2ω fields for various molecules [26,[29][30][31][32][33]. The method is capable of discriminating the head-tail order of the molecules, which is generally difficult to achieve by single-frequency laser fields. Furthermore, this method can be applied to a broad range of molecules, including the relatively large molecules that accompany heavy fragmentation [33].
In this study, we investigated the OSM-TI of allyl halides by using LP phase-controlled ω + 2ω laser fields to explore molecular TI for relatively large molecules. The geometric structure of the HOMO of four allyl halides (C 3 H 5 X; X = F, Cl, Br, I) can be systematically changed by replacing the halogen atom [31]. The mechanism of molecular TI will be discussed within the context of WFAT.

Experiment
Generally, a single-frequency LP laser field-with a waveform that is symmetric with respect to the negative and positive directions along the laser polarization-cannot discriminate the head-tail order of molecules because the ionization rate of molecules with negative orientation along the direction of polarization is equal to the rate of those with positive orientation. Meanwhile, the total electric field of an LP laser field consisting of two frequencies, the fundamental (ω) and its second harmonic (2ω), is given by where E 1 and E 2 are the amplitudes of the electric fields and φ is the relative phase difference between the fundamental and its second harmonic. The amplitude of the electric field in the positive (negative) direction is twice that in the negative (positive) direction when φ = 0 (π ) and E 1 = 2E 2 . When atoms and molecules are ionized by phase-controlled ω + 2ω laser fields, directionally asymmetric TI is expected to be induced. The TI rate of molecules with negative orientation is different from the rate of those with positive orientation if the relevant ionization is a nonlinear process such as TI. As a result, the asymmetric waveform of an LP ω + 2ω laser field can selectively ionize molecules while discriminating the headtail order of molecules with asymmetric structure. We have experimentally demonstrated that OSM-TI is induced as a consequence of the directionally asymmetric TI of molecules with an asymmetric HOMO [26,[29][30][31][32][33].
The experimental apparatus, which has been described previously [33], consisted of a Ti:sapphire laser system, a robust phase-controlled ω + 2ω laser field generator, and a time-of-flight mass spectrometer (TOF-MS) equipped with a supersonic molecular beam source. Briefly, the output beam of the Ti:sapphire laser system (800 nm wavelength, 130 fs duration, 1.0 mJ pulse energy, 20 Hz repetition rate) was delivered to the robust phase-controlled ω + 2ω laser field generator, which was designed so that both the ω and 2ω beams pass through almost identical paths [33]. This generator enabled us to provide stable phase-controlled ω + 2ω laser pulses and to control φ between the ω and 2ω pulses by using a rotatable 1 mm-thick quartz plate with a resolution of about 30 as (0.05 π ) after the second-harmonic generation (β-barium borate, type I phase-matching, 1 mm thickness, conversion efficiency: 30%) [33]. The ratio I 2 /I 1 was adjusted to around 0.25 (E 2 /E 1 = 0.5), where I 1 and I 2 are the intensities of the ω and 2ω pulses, respectively. The phase-controlled ω + 2ω laser beam was focused on a supersonic molecular beam of allyl halides in the TOF-MS by an aluminium concave mirror (200 mm focal length). The TOF-MS consisted of a Wiley-McLaren type two-stage accelerator, a field-free drift tube, and an ion detector. Ions generated by the ω + 2ω laser pulses were accelerated towards the ion detector by static electric fields. After passing through a drift tube, the photofragment ions were detected by the ion detector composed of a microchannel plate. The TOF spectra of parent ions and photofragment ions were recorded by a digital oscilloscope.
We used the definition of the experimental configuration based on the polarization direction, the detection axis, and the direction of the electric field maxima at relative phase difference φ = 0 as reported previously [26]. The polarization direction of the ω + 2ω laser fields is set to be horizontal and parallel to the detection axis, and we define φ = 0 to be the condition when the electric field maximum points towards the ion detector. In this configuration, we could simultaneously measure the phase dependence of all photofragment ions at identical φ values and laser intensities. To calibrate φ, we performed simultaneous measurements using gas mixtures of target molecules and reference C 3 H 5 I molecules [34]. This method provides an accurate phase relationship between target molecules and reference molecules under identical experimental conditions, i.e., within the same experimental run. The duration of the laser pulses used in this study is 130 fs so that the 'origin' does not mean the absolute zero of φ but, for convenience's sake, within the temporal resolution of about 20 fs, and the influence of an unstable carrier-envelope phase can be neglected.
Gas samples of C 3 H 5 F were prepared by dilution (5%) with helium gas with a total pressure of 0.1 MPa. Other allyl halides were prepared by the evaporation of liquid allyl halides into helium gas with a pressure of 0.1 MPa. Figure 1 shows the molecular structures and isocontours of the HOMOs of the four allyl halide molecules as determined by ab initio calculations using the Gaussian 03 W software package [28] (method: MP2; basis sets: 6-31++G(2df,p) for C 3 H 5 F and C 3 H 5 Cl, and LanL2DZ augmented by polarization functions and diffuse functions for C 3 H 5 Br and C 3 H 5 I). The HOMOs of all the allyl halide molecules show a large amplitude around the C = C double bond. The amplitude of the wavefunction for the C = C double bond side becomes slightly smaller from C 3 H 5 F to C 3 H 5 I. On the other hand, the amplitude of the wavefunction for the halogen-atom side becomes gradually larger from C 3 H 5 F to C 3 H 5 I, reflecting the decrease of the ionization potential for the halogen atoms. The HOMOs of all the allyl halide molecules are symmetric with respect to the C = C plane (not shown). The amplitude of the wavefunction for the halogen-atom side is apparently larger than that of the C = C double bond in C 3 H 5 I, leading  to an asymmetric structure of its HOMO. The degree of asymmetry decreases gradually from C 3 H 5 I to C 3 H 5 Cl, and then apparently reverses for C 3 H 5 F. As shown in figure 1, the geometric structure of the HOMO of the four allyl halides can be systematically changed by replacing the halogen atom [31]. Figure 2 shows the TOF spectra of singly charged ions generated by the dissociative ionization of C 3 H 5 X (X = I, Br, Cl, F) with one-colour irradiation by an 800 nm pulse. We observed singly charged CH 3 + , C 2 H 3 + , C 3 H 3 + , C 3 H 5 + , X + , and parent C 3 H 5 X + ions in all the molecules. We adjusted the laser intensity in the vicinity of the regime where doubly charged fragment ions due to Coulomb explosions were observed. The minimum intensity was 2.0 × 10 12 W cm −2 for C 3 H 5 I, and the maximum intensity was 1.0 × 10 13 W cm −2 for C 3 H 5 F, reflecting the difference in ionization potentials among the molecules. The spectra for C 3 H 5 Br and C 3 H 5 Cl are split due to the presence of isotopes for Br and Cl atoms (the abundance ratios are 79 Br/ 81 Br = 50.7%/49.3% and 35 Cl/ 37 Cl = 75.8%/24.2%). Each halogen photofragment exhibited a pair of peaks, the first peak resulting from ions emitted directly towards the detector and the second from those ions that were first emitted in the backward direction before being reversed by the extraction fields. The spacing of the forward and backward peaks reflects kinetic energy release. Each halogen ion showed symmetric forward-backward peaks under onecolour irradiation.

Results and discussion
We believe that tunnelling ionization from HOMO is the dominant process. The orientation-dependent TI rate of CO molecules induced by single-colour CP laser pulses with an intensity of 4.0 × 10 14 W cm −2 have been measured by COLTRIMS [28]. The contribution of HOMO-1 to the dissociative single ionization process in that experiment was ∼30% of the total signal in the experiment, with a laser intensity of about ten times higher than that used in our experiments. Therefore, we can safely say that the contribution of HOMO-1 seems to have been much smaller in the present study.
When the molecules were irradiated with the ω + 2ω pulses, breaking of the forward-backward symmetry was clearly observed in the TOF spectra. Expanded views of typical TOF spectra of halogen ions and counterpart cations C 3 H 3 + and C 3 H 5 + with relative phase differences φ = 0 and π are shown in figure 3. In the case of C 3 H 5 F ( figure 3(a)), the forward (backward) peak of the F + ions is predominant over the backward (forward) peak at φ = 0 (π ). This result shows that F + ions were emitted preferentially towards the forward (backward) direction when the optical electric field maximum pointed in the forward (backward) direction. In the cases of C 3 H 5 Br and C 3 H 5 I (figures 3(c) and (d)), forwardbackward asymmetry was observed for Br + and I + but with a trend opposite to F + with respect to φ = 0 and π . In the case of C 3 H 5 Cl ( figure 3(b)), phase-dependent behaviour was not clearly observed in the TOF spectrum. However, the counterpart C 3 H 3 + and C 3 H 5 + cations were not split into forward-backward peaks in all the allyl halide molecules, and phase-dependent behaviour was not clearly discernible in the TOF spectra. Figure 4 shows the ratio of the forward and backward yields (I f /I b ) as a function of φ. For the C 3 H 3 + , C 3 H 5 + cations, which were not clearly split into forward-backward peaks, we obtained I f /I b from the area of the forward and backward sides with respect to the peak centre. A clear periodicity of 2π was observed in the I f /I b ratio for the halogen ions. On the other hand, we observed phase-dependent behaviour not by counterpart cations C 3 H 5 + but by C 3 H 3 + in all the allyl halide molecules. We examined why the C 3 H 5 + cations have no phase dependence in the molecules. One possible explanation is that it is due to a slow dissociation process, in which C 3 H 5 + is   produced on a time scale longer than the rotational period, allowing orientation averaging. The experimental results show that prompt dissociation, which ensures the axial recoil and conserves information regarding the molecular orientation at a moment of dissociation, accompanies the dissociation of two hydrogen atoms in addition to the halogen atom. The phase dependences between the halogen ions and the counterpart C 3 H 3 + cations were completely out of phase with each other for all the allyl halide molecules. This result shows that a phase-controlled ω + 2ω optical field discriminates the headtail order of oriented molecules.
As an additional experiment to calibrate φ and to confirm the orientation direction of the ionized molecules, we performed simultaneous measurements using gas mixtures of C 3 H 5 F-C 3 H 5 I, C 3 H 5 Cl-C 3 H 5 I, and C 3 H 5 Br-C 3 H 5 I [29,31]. The forward/backward yield ratios I F /I B of X + generated from C 3 H 5 X (X = F, Cl, Br) and I + generated from C 3 H 5 I were measured as a function of φ. The F + generated from C 3 H 5 F and the I + generated from C 3 H 5 I were completely out of phase with each other. Furthermore, the Cl + and Br + generated from C 3 H 5 Cl + and C 3 H 5 Br + and the I + generated from C 3 H 5 I were completely in phase with each other. (These results using gas mixtures were used in the calibration of φ [34], and are reflected in figure 4, in which the phase dependence of F + is a cosine type whereas those of Cl + , Br + , I + are -cosine types). These results indicate that the direction of the oriented molecules is the same in C 3 H 5 I, C 3 H 5 Br, and C 3 H 5 Cl and that the C 3 H 5 F molecule is oriented in the opposite direction from the other three allyl halides. We have experimentally succeeded in observing the transition of the trend in the direction of the ionized molecules by using a systematically changing molecular system.
We now discuss the mechanism of OSM ionization. The directions of the ionized molecules obtained from phase dependence of the forward-backward yield ratios (I f /I b ) show that (1) for C 3 H 5 F, electrons are much more likely to be extracted from the allyl part and (2) for C 3 H 5 Cl, C 3 H 5 Br, and C 3 H 5 I, in contrast, electrons are much more likely to be extracted from the halogen atom by the phase-controlled ω + 2ω field at φ = 0 (π ). For C 3 H 5 F and C 3 H 5 I, we can safely say that the directions of the detected molecules are consistent with those expected by the molecular ADK model, where electrons are extracted from the large-amplitude parts (allyl part for C 3 H 5 F and the halogen atom for C 3 H 5 I). For C 3 H 5 Cl and C 3 H 5 Br, since the direction of the ionized molecules cannot be anticipated by just looking at the shape of the HOMOs due to the intermediate structure, a comparison between experiment and calculation would be required.
Finally, we describe the most recent situation regarding the development of a theory for molecular TI. A theory for molecular TI that considers the Stark effect, WFAT has been developed within the single-active-electron approximation [24,25]. The dominance of either the Stark shift or the shape of the HOMO has been shown to depend on the molecule and cannot be determined by just looking at the shape of the HOMOs [24,25]. For one of the simplest cases of the heteronuclear diatomic molecule CO, however, the WFAT calculates maximum ionization rates that imply that the HOMO amplitude on the O-side is smaller, which is opposite to the experimental results [35]. Since the WFAT contains all the information on the TI rates from the HOMO, the main source of the difference between theory and experiment is multi-electron effects such as the dipole moment of the core cation of CO + .
A recent time-dependent Hartree-Fock calculation shows good agreement with experimental data for the circularly polarized laser field, and the importance of dynamic core polarization effects is discussed [36]. We have reported that the mechanism of the OMS-TI induced by phase-controlled ω + 2ω laser fields can be explained by the geometric structure of the HOMO [26,[29][30][31][32][33]; thus some kind of screening due to multi-electron effects that cancel the Stark shift could be induced. A comprehensive theory involving multielectron effects for any molecule is now in progress, and the experimental results of OSM-TI for C 3 H 5 Cl and C 3 H 5 Br with intermediate conditions will be a benchmark for the developed theory of molecular TI.

Conclusions
We have investigated the orientation-sensitive molecular TI induced by intense phase-controlled ω + 2ω pulses for the case of systematically changing orbital asymmetry C 3 H 5 X (X = F, Cl, Br, I). The ω + 2ω method is applicable to a broad range of molecules that show hard fragmentation processes. The orientation direction of the ionized molecules is the same for C 3 H 5 I, C 3 H 5 Br, and C 3 H 5 Cl, and is in the opposite direction for C 3 H 5 F. In the cases of HOMOs with large asymmetry, C 3 H 5 F and C 3 H 5 I, the direction of the ionized molecules is consistent with that expected by OSM-TI based on the molecular ADK model, and the effect of the geometric structure of the HOMO is stronger than of the Stark shift. In the cases of C 3 H 5 Cl and C 3 H 5 Br, for which it is not so easy to anticipate the direction of the ionized molecules just by looking at the shape of the HOMOs with intermediate structure, the Stark effect might make some contribution. Although the WFAT, which fully contains the effects of the geometric structure and the Stark shift of the HOMO, has been developed within the singleactive-electron approximation [35], it has been pointed out that a comprehensive theory involving multi-electron effects for molecules is required [36]. Since our previous experiments have shown that the mechanism of the OMS-TI induced by the a phase-controlled ω + 2ω laser fields can be explained by the geometric structure of the HOMO [26,[29][30][31][32][33], some kind of screening due to multi-electron effects that cancel the Stark shift could be induced. Our experimental results of OSM-TI for C 3 H 5 Cl and C 3 H 5 Br with intermediate conditions will be a benchmark for the developed theory of molecular TI. Moreover, as it has been reported experimentally that the molecular TI process is dependent on laser polarization [37], the accumulation of experimental results for various molecules investigated by different methods such as cold target recoil ion momentum spectroscopy [28] and the TI for oriented molecules by static electric fields [21] is required for in-depth understanding of molecular TI.


extfieldsofstudy: Chemistry Medicine

text:
Synthesis and characterization of Schiff's bases of sulfamethoxazole

Background Schiff's bases are excellent ligands which are synthesized from the condensation of primary amines with carbonyl groups. Findings The classical reaction for the synthesis of Schiff's bases in an ethanolic solution and glacial acetic acid as a catalyst was followed in the synthesis of substituted sulfamethoxazole compounds. Conclusions Some Schiff's bases containing sulfamethoxazole nucleus have been synthesized and characterized. The present compounds are hoped to be applied in the photostability of PVC.

Background
Schiff's bases are an important class of organic compounds [1]. They were first reported by Hugo Schiff in 1864 [2]. Schiff's bases are condensation products of primary amines with carbonyl compounds. The common structural feature of these compounds is the azomethine group with the general formula RHC = N-R 1 , where R and R 1 are alkyl, aryl, cycloalkyl, or heterocyclic groups [1]. Structurally, a Schiff's base (also known as imine or azomethine) is a nitrogen analogue of an aldehyde or ketone in which the carbonyl group (>C = O) is replaced by an imine or azomethine group. Schiff's bases have also been shown to exhibit a broad range of biological activities, including antifungal, antibacterial, antimalarial, antiproliferative, anti-inflammatory, antiviral, and antipyretic properties [3,4]. Imine or azomethine groups are present in various natural, naturally derived, and nonnatural compounds. The imine group present in such compounds has been shown to be critical to their biological activities [5][6][7]. Schiff's bases are important compounds owing to their wide range of industrial applications [8]. Schiff's bases are used in the photostabilization of poly (vinyl chloride) polymers against photodegradation by ultraviolet radiation [9][10][11] and are also used to improve poly(methyl methacrylate) from degradation [12] and to prevent polystyrene from photodegradation by their addition to polymer films [13,14].

Methods
Fourier transform infrared (FTIR) spectra were registered on a SHIMADZU (8300, Kyoto, Japan) infrared spectrophotometer, using KBr discs. Proton nuclear magnetic resonance ( 1 H-NMR; 600 MHz) spectra were obtained at room temperature with Bruker equipment (Madison, WI, USA) using TMS as an internal standard in dimethyl sulfoxide (DMSO). Melting points were recorded using hot-stage Gallenkamp melting point apparatus (Loughborough, UK) and were uncorrected. Analytical grade chemicals (BDH, G.C.C., Hopkin & William Corporation, Poole, UK) were used throughout the project.

Results and discussion
The synthesis of Schiff's bases with different specific aldehydes in ethanol as a solvent and catalyst (glacial acetic acid) resulted in five new series of Schiff's bases with the general formula RHC = N-R 1 . Here R 1 = sulfamethoxazole and R = benzaldehyde, 4-bromobenzaldehyde, 2-hydroxybenzaldehyde (salicylaldehyde), 4-N,N-dimethylbenzaldehyde, and 3-nitrobenzaldehyde were synthesized by the reaction of sulfamethoxazole and substituted aldehydes in ethanol (Scheme 1). Such compounds were characterized by different physicochemical techniques like melting point, elemental analysis, FTIR spectroscopy, and multinuclear NMR ( 1 H).

Physical properties of the prepared Schiff's bases
The physical properties of sulfamethoxazole and its derivatives including melting point, color, and elemental analysis are tabulated in Table 1.
The structure of the prepared Schiff's bases was confirmed by infrared spectroscopy. The FTIR spectra of sulfamethoxazole and its prepared compounds, as shown in Table 2, showed that the band of NH 2 was found in sulfamethoxazole in the location 3,298 cm −1 and then vanished. After that, the band of NH appeared in the prepared Schiff's bases with different shifting from 3,250 to 3,287 cm −1 . The band of C = N for imine stretching vibration was also not found in sulfamethoxazole, and it Ethanol/ few drops of glacial acetic acid as catalyst appeared in the prepared Schiff's bases with shifting from 1,603 to 1,650 cm −1 . Also, the band of C = N for ring stretching vibration shifted from 1,620 cm −1 in sulfamethoxazole to 1,615 to 1,630 cm −1 in the prepared compounds.
The ultraviolet-visible spectrophotometry technique is used to characterize sulfamethoxazole and its derivatives in DMSO as a solvent. The ultraviolet-visible electronic spectra of the prepared Schiff's bases showed absorption bands that could be attributed to π → π* electronic transitions; these transitions are assigned in relevance to the structures of the compounds. The electronic spectrum of sulfamethoxazole shows a band at the wavelength 280 nm; this may be attributed to the π → π* electronic transition. In the prepared Schiff's bases, the bands shifted to wavelengths different from that of the corresponding band in sulfamethoxazole, as shown in Table 3, which appear in the wavelength range between 270 and 360 nm. These transitions may be attributed to π → π* and n → π* electronic transitions.
The 1 H-NMR spectrum of compound (1) showed the following characteristic chemical shifts (DMSO as a solvent): the singlet signal at δ = 2.212 ppm suggested the attribution of the protons of the CH 3 group, the singlet signal at δ = 6.029 ppm suggested the attribution of the proton of CH of the isoxazole ring, the multiplet signal at δ = 6.743 to 7.768 ppm suggested the attribution of the protons of two aromatic benzene rings, the singlet signal at δ = 8.764 ppm suggested the attribution of the proton of the CH = N group, the singlet signal at δ = 9.352 ppm suggested the attribution of the proton of the NH group, and the singlet signal at δ = 10.525 ppm suggested the attribution of the proton of the OH group.
Also, the 1 H-NMR spectrum of compound (5) showed the following characteristic chemical shifts (DMSO as a solvent): the singlet signal at δ = 2.200 ppm suggested the attribution of the protons of the CH 3 group, the singlet signal at δ = 5.833 ppm suggested the attribution of the proton of CH of the isoxazole ring, the multiplet signal at δ = 6.789 to 7.749 ppm suggested the attribution of the protons of two aromatic benzene rings, the singlet signal at δ = 8.698 ppm suggested the attribution of the proton of the CH = N group, and the singlet signal at δ = 9.602 ppm suggested the attribution of the proton of the NH group [15], as shown in Table 4.