extfieldsofstudy: Chemistry Medicine

text:
A quantitative structure- property relationship of gas chromatographic/mass spectrometric retention data of 85 volatile organic compounds as air pollutant materials by multivariate methods

A quantitative structure-property relationship (QSPR) study is suggested for the prediction of retention times of volatile organic compounds. Various kinds of molecular descriptors were calculated to represent the molecular structure of compounds. Modeling of retention times of these compounds as a function of the theoretically derived descriptors was established by multiple linear regression (MLR) and artificial neural network (ANN). The stepwise regression was used for the selection of the variables which gives the best-fitted models. After variable selection ANN, MLR methods were used with leave-one-out cross validation for building the regression models. The prediction results are in very good agreement with the experimental values. MLR as the linear regression method shows good ability in the prediction of the retention times of the prediction set. This provided a new and effective method for predicting the chromatography retention index for the volatile organic compounds.

Introduction
Volatile organic compounds (VOCs) are molecules that have a high vapor pressure and low water solubility, like organic chemicals in general. There are many different compounds which may be classified as volatile organic compounds (VOCs). The compounds the nose detects as smells are generally VOCs. Many VOCs are humanmade chemicals that are used and produced in the manufacture of paints, pharmaceuticals, and refrigerants. VOCs typically are industrial solvents, such as trichloroethylene; fuel oxygenates, such as methyl tert-butyl ether (MTBE), or by-products produced by chlorination in water treatment, such as chloroform.
VOCs are common ground-water contaminants. Many volatile organic compounds are also hazardous air pollutants. VOCs also play a major role in the formation of various secondary pollutants through photochemical reactions in the presence of sunlight and nitrogen oxides. Furthermore, some VOCs could contribute to the atmospheric ozone depletion and the build-up persistent pollutions in remote areas. Therefore, these compounds have been an important environmental issue over the last two decades and have attracted significant attention from different research groups. Although scientist are provided a sensitive and specific analytical method for identifying and measuring the VOCs [1][2][3][4][5], but there are some limitations for these methods. For example, sorbents are normally selective in, although not limited to, adsorbing/ absorbing certain classes of VOCs or sometimes high sample volumes of air are needed for identification or measuring the VOCs. Besides the above mentioned method, the experimental determination of retention time is time-consuming and expensive. Alternatively, quantitative structure-retention relationship (QSRR) provides a promising method for the estimation of retention time based on descriptors derived solely from the molecular structure to fit experimental data [6][7][8]. A QSRR study involves the prediction of chromatographic retention parameters using molecular structure. QSRR studies are widely investigated in gas chromatography (GC) [9][10][11] and high-performance liquid chromatography (HPLC) [12]. The chromatographic parameters are expected to be proportional to a free energy change that is related to the solute distribution on the column.
Model development in QSAR/QSPR studies comprises different critical steps as (1) descriptor generation, (2) data splitting to calibration (or training) and prediction (or validation) sets, (3) variable selection, (4) finding appropriate model between selected variables and activity/property and (5) model validation.
In the present work, a QSRR study has been carried out on the GC/MS system retention times (t R' s) for 85 volatile organic compounds by using structural molecular descriptors. We applied a linear MLR and a nonlinear algorithms ANN along with stepwise regression as variable selection method.

Computer hardware and software
All calculations were run on a Toshiba personal computer with a Pentium IV as CPU and windows XP as operating system. The molecular structures of data set were sketched using ChemDraw (Ver. 11, supplied by Cambridge Software Company). The sketched structures were exported to Chem3D module in order to create their 3D structures. Energy minimization was performed using MM+ molecular mechanics and AM1 semi-empirical to obtain the root mean square (RMS) gradient below 0.01 kcal/mol Å. The next step in developing a model is generation of the corresponding numerical descriptors of the molecular structures. Molecular descriptors define the molecular structure and physicochemical properties of molecules by a single number. A wide variety of descriptors have been reported for using in QSAR/QSPR analysis. Here, 477 descriptors were generated for each compound, using Dragon (ver. 3

Data set
The retention times of volatile organic compounds have been obtained from reference [13], we have also added the 8 compounds (1, 4-difluorobenzene, chlorobenzene,1,4-dichlorobenzene,4-bromofluorobenzene, 1,2-dichlorobenzene, dichloroethane, toluene, fluorobenzene) that are generally used as internal standards in the GC/MS analysis. All 85 VOCs are listed in Table 1. The analytes have been extracted from the matrices and components are separated, identified and measured by a wide bore capillary column of a gas chromatography/mass spectrometer detector system.

Principal component analysis
Principal components analysis (PCA) was performed on the calculated structural descriptors to the whole data set of VOCs (Table 1), for investigation the distribution in the chemical space, which shows the spatial location of samples to assist the separation of the data into training and test sets [14]. The PCA results show that two principal components (PC1 and PC2) describe 59.7% of the overall variances: PC1 =35.38%, PC2= 24.31% ( Figure 1). Since almost all variables can be accounted for the first two PCs, their score plot is a reliable representation of the spatial distribution of the points for the data set. The plot of PC2 against PC1, Figure1, displays the distribution of compounds over the first two principal components space. According to the results of PCA, 2 molecules were removed as outliers( Figure 1) the remaining molecules were divided into a 67 compounds as training set to develop the models and 16 compounds as prediction set to evaluate the models.
Descriptor selection 477 descriptors were calculated by Dragon for each molecule, therefore we have 85×447 data matrix X. The rows and columns of this matrix are the number of molecules and molecular descriptors respectively. All descriptors have been checked to ensure: (a) that values of each descriptor are available for each structure (the investigation of missing values) and (b) that there is a significant variation in these values over studied molecules. Descriptors which has zero value for each structure or shows constant values over molecules in the under study data set are discarded. X and y(dependent variable) were preprocessed by autoscaling. We used stepwise regression [15] for variable selection. PCA [16] is applied for reduction of the descriptor space (variable space).
In the variable selection step, feature selection (FS) [17][18][19] and feature extraction (FE) are commonly used methods to handle a large number of calculated descriptors in QSAR/QSPR studies. In FE methods, by the use of principal component analysis (PCA), the information contained in the descriptors data matrix is extracted into new orthogonal variables with lower dimensions.
In stepwise procedure a variable that entered the model in the earlier stages of selection may be deleted at the later stages. The calculations made for inclusion and elimination of variables are the same as forward selection and backward procedures. That is, the stepwise methods are essentially a forward selection procedure, but at each stage the possibility of deleting a variable, as in backward elimination, are considered. Stepwise addition of further descriptors is continued to find the best multi-parameter regression models with the optimum values of statistical criteria (highest values of R 2 and the cross-validated R 2 cv ). With stepwise regression 23 descriptors were selected and a simple ''break point'' technique was used to control the model expansion in Table 1 The data set and the corresponding experimental and predicted t R values by MLR and ANN methods. the improvement of the statistical quality of the model, by analyzing the plot of the number of descriptors involved in the obtained models versus squared correlation coefficient values corresponding to those models ( Figure 2). The model corresponding to the break point is considered the best optimum model, in this way 6 descriptors that have high contribution in the variance of dependent variable (t R ) were selected and used to build the models.

Multivariate regression models
Multiple linear regression (MLR), is the most frequently used modeling method in QSAR/QSPRs. MLR yields models that are simpler and easier to interpret than factor based methods like PCR(principal component regression) [20,21] and PLS(partial least squares) [22][23][24][25][26][27]. However, due to the co-linearity between structural descriptors, MLR is not able to extract useful information from structural data, and over fitting problem is encountered. ANN model used to handle the probable nonlinear relationship between descriptors and retention times. One of the most striking point of ANN is that its free from collinearity problem.
We have tested the stability of models by random selection of train and test sets to assure the resulting models are not just chance correlations. 100 models by random selection are constructed and the average of R 2 and RMSEC are 0.971 and 0.162, respectively. The results show that the different selected test sets from the data have very similar R 2 values. This indicates that the final model is reliable and has a satisfactory stability.

Multiple linear Regression (MLR)
Multiple linear regression [28] are the most widely used and known modeling methods.
MLR is performed either to study the relationship between the response variable and predictor variables or to predict the response variable based on the predictor variables. The validation of the model was performed by cross-validation method. With cross-validation, one sample was kept out (leave-one-out) of the calibration and used for prediction. The process was repeated so that each of the samples was kept out once. The predicted values of left-out samples were then compared to the observed values using prediction error sum of squares(PRESS) which indicating the residuals are computed in cross-validation. PRESS eq. (1) and RMSE, root mean squared error, eq. (2) are defined as: where ŷ i is the estimated value of the ith object , y i is the corresponding reference value of this object and N is the number of the objects. Table 2 shows the statistical parameters of the model corresponding to the 6 independent variables. The unstandardized coefficients and standard error that allows the comparison of the relative weight of the variables in the model are presented in Table 3. Plots of experimental t R values and residuals (experimental t R -predicted t R ) versus predicted t R values, obtained by the MLR modeling, are shown in Figure 3 The agreement observed between the predicted experimental values in and the random distribution of residuals about zero mean confirms the good predictive ability of MLR modeling.
To show the absence of correlation by chance between independent and dependents variables randomization test was performed. The MLR modeling was performed on the randomized data, the elements of the response vector were randomly mixed and the modeling was performed on the matrix X and new response vector [29]. The squared correlation coefficient of the model with the randomly selected data (R 2 CR ) was much lower than those of the original model, it could be considered that the model was reliable, and had not been obtained by chance. The low mean of correlation value (R 2 CR =0.091) obtained by chance confirms this result.
One of the most important problems in QSAR/QSPR analysis is establishing the domain of applicability for each model [30]. In the absence of the applicability domain restriction, each model can formally predict the property of any compound, even with a completely different structure from those included in the training set. The analysis of the chemical applicability domain (AD) of the obtained model and the reliability of the predictions are verified by the leverage approach, which is based in computing the leverage, h*, for each compound for which the QSPR model is used to predict the property under study. The warning leverage is generally fixed at 3k/n, k being the number of model parameters and n being the number of training set compounds (Fig. 4).

Artificial neural network
Artificial neural networks (ANNs) [31][32][33] are biologically inspired algorithms designed to simulate the way in which the human brain processes information. ANNs are parallel computational models which are able, at least in principle, to map any nonlinear functional relationship between an input and an output hyperspace to the desired accuracy. The network receives a set of inputs, which are multiplied by each neuron's weight. These products are summed for each neuron, and a nonlinear transfer function is applied. A set of six descriptors that were appeared in the MLR model, were used as input parameter of the network.
In this investigation, the logsig function was used as a transfer function of hidden layer, traingdx function as a training function and a linear function for the output layer. We optimized the parameters such as number of nodes, momentum (a) and learning rate (h). These values were obtained during the network training. The initial weights were randomly selected between (-0.3 and 0.3). A neural network with 6-6-1 topology was developed with optimum momentum and learning rate.
Stepwise MLR is a simple and powerful method to modeling the linear correlation of significant input variables but it does not account for nonlinear relationship, so ANN model was constructed for this purpose. To compare the performance of the MLR and ANN models descriptors that used in the MLR model should be the same as the input variables for generation the network. The parameters and results of these models are shown in Table 4. As it is obvious from the results in Table 4 ANN has more or less same efficiency as well as MLR to map the relationship between input objects and response values.

Description of models descriptors
Molecular descriptors will probably play an increasing role in scientific growth. In fact, the availability of large numbers of theoretical descriptors containing diverse source of chemical information would be useful to better understand relationship between molecular structure and experimental evidence.
The X1sol (solvation connectivity index) and X5A (average connectivity index) appearing in the MLR model mainly show the topological characteristics.
where L a is the principal quantum number (2 for C, N, O atoms,3 for Si, S, Cl, …)of the a th atom in the k th subgraph and δ a the corresponding vertex degree; k is the total number of m th order subgrah; n is the number of vertices in subgraph. The normalization factor 1/ (2 m+1 ) is defined in such a way that the indices m χ and m χ s for compounds containing only second-row atom coincide. The X1sol shows a regression coefficient (0.823), which is the largest among the descriptors appearing in the model. This parameter can be considered as entropy of solvation and somehow indicates the dispersion interactions occurring in the solutions. X5A also is a measure of branching of the molecules. The large contributions of this parameter in the retention behavior of VOCs are in agreement with the contribution that one would expect for the interaction of a stationary phase with the VOCs. X5A is the average connectivity index that can be obtained from molecular graph.
MATS2m (Moran autocorrelation− lag 2/weighted by atomic masses) and ATS2m (broto-moreau autocorrelation of a topological structure-log2/weighted by atomic masses) are 2D autocorrelation descriptors [34], which are also obtained from molecular graphs, by summing the products of atom weights of the terminal atoms of all the paths of the considered path length (the lag). The MATS2m and ATS2m are related to the atomic property of a molecule, these descriptors concerning molecular size, which influence retention of compound. The term ATS2m is a graph invariant describing now the considered property (retention time) is distributed along the topological structure. Molecular descriptors based on the autocorrelation function AC l defined as: where f(x) is any function of the variable x and l is the lag representing an interval of x; a and b defined the total studied interval of the function. The function f(x) is usually a time dependent function such s a timedependent electrical signal, or spatial dependent function such as the population density in space.
Mor21m (3D MoRSE-signal 21/weighted by atomic masses) and Mor10m (3D MoRSE-signal 10/weighted by atomic masses) are among the 3D-MoRSE descriptors. 3D-MoRSE [35] descriptors (Mor21m, Mor10m) are molecule atom projections along different angles, such as in electron diffraction. They represent different views of the whole molecule structure, although their   meaning remains not too clear. 3D-MoRSE descriptors are based on the idea of obtaining information from the 3D atomic coordinates by the transform used in electron diffraction studies for preparing theoretical scattering curves. 3D-MoRSE descriptors are important because they take into account the 3D arrangement of the atoms without ambiguities (in contrast with those coming from chemical graphs), and also because they do not depend on the molecular size, thus being applicable to a large number of molecules with great structural variance and being a characteristic common to all of them. All of these descriptors are individually important to increase or decrease the retention times but the final effect is not result in each descriptor individually and all of the selected descriptors as a group can influenced on the dependent variable. This problem in many cases led to the hard interpretation of the model.

Conclusion
QSRR analysis was performed on a series of volatile organic compounds, for prediction the retention times.
Stepwise regression was used to as variable selection method. The comparison of the models that were developed by selected descriptors shows that MLR can present the satisfactory result for predicting of retention time of VOCs. Comparison of the linear (MLR) and nonlinear (ANN) methods showed the little superiority of the ANN over the MLR model for the prediction of the retention time of VOCs, so the results suggest that a relation between the molecular descriptor and the retention times is linear. The QSRR models proposed with the simply calculated molecular descriptors can be used to estimate the chromatographic retention times of new compound even in the absence of the standard candidates. The most important selected descriptors are topological which can capture the variance in the retention times what related to the size and shape of the molecules.


s2fieldsofstudy: Chemistry Physics

text:
Electronic structure of cubic ScF$_3$ from first-principles calculations

The first-principles calculations have been performed to investigate the ground state properties of cubic scandium trifluoride (ScF$_3$) perovskite. Using modified hybrid exchange-correlation functionals within the density functional theory (DFT) we have comprehensively compared the electronic properties of ScF$_3$ obtained by means of the linear combination of atomic orbitals (LCAO) and projector augmented-waves (PAW) methods. Both methods allowed us to reproduce the lattice constant experimentally observed in cubic ScF$_3$ at low temperatures and predict its electronic structure in good agreement with known experimental valence-band photoelectron and F 1$s$ X-ray absorption spectra.

Introduction
Scandium trifluoride (ScF 3 ) is a promising material attracted much attention due to recently discovered strong negative thermal expansion (NTE) coefficient over a wide range of temperatures from 10 to 1100 K [1].
At atmospheric pressure ScF 3 has cubic (space group P m3m) ReO 3 -type structure ( Fig. 1) down to at least 10 K [1]. However, opposite to the metallic ReO 3 , which has very week NTE effect [2,3], scandium trifluoride is an insulator with band gap of more than 8 eV [4]. A cubicto-rhombohedral phase transition occurs in ScF 3 at high pressure (P >0.5 GPa at T ∼300 K or P =0.1-0.2 GPa at 50 K) as determined from X-ray and neutron diffraction studies [1,5]. Raman spectroscopy confirms that upon pressure increase cubic ScF 3 undergo a phase transition to the rhombohedral (space group R3c) phase [5,6].
The electronic structure of ScF 3 thin films has been investigated by means of resonant photoemission spectroscopy at the Sc 2p and F 1s absorption edges in Ref. [4]. Two chargetransfer-type satellites, the first one at about 13 eV below the main peaks in the valence-band and the second one with an energy separation of about 9-10 eV, were observed and indicate the strong hybridization effect between Sc 3d and F 2p states [4].
The phonon properties of ScF 3 have been studied using a combination of inelastic neutron scattering experiment with ab initio calculations of lattice dynamics [7]. It was shown that a description of NTE within the quasi-harmonic approximation is not reliable in the case of ScF 3 . The authors in [7] have demonstrated that the R4+ mode (the one with the lowest energy at R-point of Brillouin zone (BZ)) has quartic potential and proposed the mechanism for NTE based on this anharmonicity. The transition between the ground state and the first excited state (∼19 meV) of the R4+ mode has been associated with experimentally observed phonon peak (∼25 meV), which stiffens upon increasing temperature [7].
In spite of mainly experimental efforts have been performed to understand the NTE in ScF 3 , its origin is still under debate. Therefore, comprehensive theoretical study is necessary to elucidate the nature of this peculiar property. In this study we report the results of ab initio electronic structure calculations for cubic ScF 3 using the linear combination of atomic orbitals (LCAO) and projector augmented-waves (PAW) methods.

Computational details
In this study ScF 3 is modeled by means of two different methods: (i) LCAO within the framework of hybrid density functional approach and (ii) PAW calculations using the generalized gradient approximation (GGA) density functional.
To perform hybrid LCAO calculations, we used the periodic CRYSTAL09 code [8], which employs Gaussian-type functions centered on atomic nuclei as the basis set for expansion of the crystalline orbitals. The full-electron basis sets used in this study for F and Sc were taken from CRYSTAL basis set library [8]. Threshold parameters for evaluation of different types of bielectronic integrals such as overlap and penetration tolerances for Coulomb integrals, overlap tolerance for exchange integrals and pseudo-overlap tolerances for exchange integral series [8] have been set to 10 −8 , 10 −8 , 10 −8 , 10 −8 and 10 −16 , respectively. Calculations are considered as converged when the total energy obtained in the self-consistent field procedure differs by less than 10 −10 a.u. in the two successive cycles.
LCAO calculations of ScF 3 have been performed using a set of hybrid Hartree-Fock/Kohn-Sham (HF/KS) exchange-correlation functionals [9,10,11,12,13] combining exact HF nonlocal exchange and KS exchange operator within the GGA as implemented in the CRYSTAL09 code [8]. We have constructed the following hybrid functionals: "BECKE & LYP", "BECKE & PWGGA", "PBE & PBE", "PBEsol & PBEsol", where the first terms refer to the KS exchange and the second terms to the KS correlation parts of a hybrid functional, while the weight of the HF exchange has been varied for each functional separately between 0 and 100%.
DFT calculations using the PAW method as implemented in the VASP code [14] were performed for comparison. We have compared PW91 [13] and PBE [9] exchange-correlation potentials as well as large-core effective core potential (LC-ECP) with three 3d 1 4s 2 valence electrons and small-core effective core potential (SC-ECP) with eleven 3s 2 3p 6 3d 1 4s 2 valence electrons for Sc atom. Fluorine atom for all calculations was described as ECP with 2s 2 2p 5 valence shell. Hybrid HF-DFT calculations have been also performed with the weigth of exact HF exchange part equal to 25%. The optimal cut-off energy has been equal to 1000 eV.
To provide the balanced summation over the direct and reciprocal lattices of ScF 3 in both LCAO and PAW methods, the reciprocal space integration has been performed by sampling the BZ with the 8×8×8 Pack-Monkhorst k-mesh [15] that results in 35 eventually distributed k-points in the irreducible BZ.

Choice of exchange-correlation functional
The influence of the exchange-correlation functional type and the weight (w HF) of exact HF nonlocal exchange in hybrid density calculations within the LCAO method on the lattice parameter in ScF 3 is shown in Fig. 2. We found that pure GGA functionals overestimate the experimental value of lattice constant a 0 =4.026Å [1], while stepwise increase of the w HF for about 10% leads to proportional decrease of a 0 by approximately −0.01Å. The PBEsol functional with w HF=18% reproduces perfectly the experimental lattice parameter and has been selected for further calculations. Values of the lattice constant a 0 obtained using pure GGA calculations by the VASP code [14] are in the range of 4.02-4.03Å if LC-ECP is used for Sc, while a 0 of 4.06-4.07Å has been obtained if SC-ECP has been adopted for Sc. Using hybrid HF-DFT calculations with the PBE0 functional [14]), the value of a 0 is equal to ∼4.04-4.05Å: it is larger when LC-ECP is used for Sc and is smaller when SC-ECP is used.
To additionally justify the choice of hybrid functional, we have calculated the value of the band gap (E g ). Until now the band gap of ScF 3 was not precisely measured experimentally, but it is estimated to be larger than 7-8 eV [4]. All hybrid functionals constructed for LCAO method (wHF corresponds to a 0 =4.026Å, see Fig. 2) show that ScF 3 is an insulator with relatively large band gap E g > 8 eV. In the PAW GGA calculations the band gap is underestimated as it is predicted by the theory for pure GGA functionals: it is equal to 5.5-6.0 eV. Hybrid PAW calculations allowed us to obtain E g equal to 8-9 eV, which is closer to the experimental estimate.
Thus, for electronic structure calculations we have finally chosen the "PBEsol & PBEsol" functional with wHF=18%, giving E g =9.8 eV, for LCAO method and the PW91 functional with wHF=25%, giving E g =8.9 eV, for PAW method.

Electronic properties
The effective charges of scandium and fluorine atoms were calculated using Mulliken population analysis [8] in LCAO method and Bader topological analysis [16] in PAW calculations. The LCAO charges are equal to q Sc =+2.28e and q F =−0.76e. In PAW method the atom charges were slightly different for two Sc pseudopotentials: q Sc =+2.70e and q F =−0.90e for LC-ECP and Figure 3. Plots of the charge density difference in (100) plane between the full charge density of crystal and electronic density of non-interacting atoms (left panel) and ions (right panel) obtained using "PBEsol & PBEsol" functional with wHF = 18%. Solid (red) and dashed (blue) isolines correspond to positive (excess) and negative (deficiency) electron density difference, respectively.  [17] and F 1s Xray absorption spectra [4]. The amplitude of the DOS in the conduction band was multiplied by a factor of 5. Energy scale is given with respect to the calculated top of the valence band. Experimental spectra has been aligned to match best the calculations.
q Sc =+2.17e and q F =−0.73e for SC-ECP. These results demonstrate a considerable deviation of effective atomic charges from formal charges caused by partly covalent nature of Sc-F bonds. Fig. 3 shows electron density maps in (100) plane of ScF 3 obtained using LCAO calculation as a difference between the full charge density of crystal and electronic density of non-interacting atoms or ions. The anisotropy of electron density induced by bond covalent contribution is clearly visible in both graphs: the electron density is larger along the crystallographic axes.
In Fig. 4 we compare the partial F p density of states (DOS) calculated by both LCAO and PAW methods with the experimental valence-band (VB) photoelectron spectrum taken from Ref. [17] and the F 1s X-ray absorption spectrum taken from Ref. [4]. The VB photoelectron spectrum of ScF 3 has a complex shape composed of the main peak and the shoulder located at the left side, which originate due to the valence band splitting upon hybridization of the F 2p and Sc 3d states [17]. Our theoretical calculations confirm such interpretation and reproduce well the width and asymmetric shape of the valence band. The F 1s absorption spectrum [4] consists of three well visible peaks at 9.3, 11.4 and 18.2 eV due to transitions from the F 1s core state to the F np states in the conduction band and above. The first two peaks correspond to the F 2p states in the conduction band, which are hybridized with the Sc 3d states. The two peaks originate from the t 2g -e g splitting of the Sc 3d states by the octahedral crystal field. The third peak at 18.2 eV corresponds to the interaction of the F 2p and Sc 4p states. Such interpretation is fully supported by the calculated F p DOS: in fact, good agreement with the F 1s absorption spectrum indicates that a relaxation of the electron states due to the F 1s core-hole is weak in ScF 3 .

Conclusions
The lattice constant, band gap and electronic properties of cubic ScF 3 were calculated using LCAO and PAW DFT computational methods. It was shown that a variation of exact HF exchange part allowed us to reproduce experimental value of the lattice constant a 0 =4.026Å [1].
The band gap obtained in hybrid HF-DFT calculations is equal to 8-10 eV and agree well with the expected value [4]. A deviation of atom charges from formal ones and an anisotropy of electronic density point to the covalent nature of Sc-F bonds in ScF 3 . Calculated partial F p DOS is in a good agreement with valence-band photoelectron [17] and F 1s X-ray absorption [4] spectra.
Good agreement between the theory and available experimental data makes promising the application of the present approach to the investigation of phonon properties in ScF 3 in the future work.


s2fieldsofstudy: Chemistry Biology

text:
An LC/MS Assay for Analysis of Vorinostat in Rat Serum and Urine: Application to a Pre-Clinical Pharmacokinetic Study

The utility of currently reported liquid chromatography mass spectrometry methods for quantitation of vorinostat in plasma or serum may be limited. These methods employ time consuming and expensive procedures such as solid phase extraction or limited-access techniques such as high turbulence liquid chromatography coupled with column switching. This study offers a simple isocratic LC/MS method validated for determination of vorinostat in both rat serum and urine. Proteins were precipitated from serum using ice-cold acetonitrile and daidzein was used as internal standard. Separation was achieved using a Phenomenex® Luna® C18 (2) (5 μm, 250 x 4.60 mm) column and isocratic elution with a mobile phase consisting of acetonitrile, water, and formic acid (30:70:0.1, v/v/v). Mass spectrometry detection using electrospray positive-mode ionization with selected ion monitoring detection was employed. The calibration curves were linear ranging from 0.05 to 50 μg/ml in serum and from 0.5 to 25 μg/ml in urine. In both biological matrices, assay precision was <15% (RSD) and the intraand inter-run bias were within acceptable range (–15% to 15%). No degradation of vorinostat in either matrix was found following three freeze-thaw cycles and 24 h storage in the auto-injector. The lower limit of quantitation in rat serum and urine were 0.05 and 0.5 μg/ml, respectively. The upper limit of quantitation in rat serum and urine were 50 and 25 μg/ml, respectively. This assay was applied successfully to a pre-clinical study of vorinostat pharmacokinetics in rats.

Introduction
Histone deacetylase (HDAC) is an enzyme that removes acetyl groups from lysine residues of proteins, including histones and transcription factors [1]. Certain cancers overexpress HDACs resulting in over-compaction of the histone-DNA complex and repression of gene transcription for an array of genes including those for cell-cycle control, apoptosis, and tumor suppression [2]. HDAC inhibitors are currently under clinical evaluation for chemotherapeutic monotherapy [3,4] and as an adjunct treatment with existing agents [5,6].
In the biomedical literature, a small number of liquid chromatography mass spectrometry (LC/MS) methods for quantitation of vorinostat in plasma or serum are reported. To our knowledge, only one validated LC/MS/MS method of vorinostat in both human serum and urine is reported [9], however, it utilizes high turbulence liquid chromatography coupled with limited-access column switching technol-ogy. Other methods including that by Kelly et al. [10] have reported an LC/MS method for determination of vorinostat in human plasma [10]. In this method, solid phase extraction (SPE) of human plasma was performed which is time consuming and expensive. In addition, use of human plasma has been shown to cause stability problems of vorinostat [9]. Many of the other reported methods of vorinostat analysis utilized tandem mass spectrometry detection, which may limit their utility as access to a MS/MS system is often limited [11,12]. Additionally, to our knowledge, few methods for analysis of vorinostat in rat serum or plasma are reported in the literature. In one method, analysis of vorinostat in rat plasma was conducted using LC-MS/MS with electrospray ionization with lower and upper limits of quantitation (LOQ) of 1 ng/ml and 2000 ng/ml, respectively [13]. Another LC/MS analysis of vorinostat in rat serum using multiple reaction monitoring (MRM) detection mode was developed with lower and upper LOQ of 5 ng/ml and 5000 ng/ml, respectively [14]. Both methods employed gradient elution and were validated for determination of vorinostat in rat serum only with relatively low upper limit LOQ.
Development of parenteral formulations of vorinostat has been hindered by its low water solubility, approximately 0.2 mg/ml, [15]. Also, pharmacokinetics of vorinostat are not optimal [2,13,14,16]. Because of this, new formulations of vorinostat for oral and intravenous administration are in development in our laboratory to improve the solubility and pharmacokinetics of vorinostat. These formulations can drastically improve solubility of hydrophobic compounds thereby greatly altering their pharmacokinetics. Appropriately, preliminary pharmacokinetic studies of these formulations indicated serum levels of vorinostat were much higher than the upper LOQ of the reported assays. Therefore, a simple isocratic LC/MS assay was developed and validated over a concentration range with lower and upper LOQ facilitating quantitation of vorinostat in both rat serum and urine following oral and intravenous administrations. In addition, the application of this LC/MS to a pre-clinical study of vorinostat pharmacokinetics in rats was investigated and findings were compared to previously reported data.

Chemicals and reagents
Vorinostat and daidzein were purchased from LC laboratories (Woburn, MA, USA). HPLC-grade acetonitrile, water, and analyticalgrade formic acid and polyethylene glycol 400 were purchased from J.T. Baker (Philipsburg, NJ, USA). Methylcellulose was purchased from Sigma-Aldrich (St. Louis, MO, USA). Male Sprague-Dawley rats (≈0.29 kg) were obtained from Simonsen Labs (Gilroy, CA, USA). Ethics approval for animal experiments was obtained from the Institutional Animal Care and Use Committee of Washington State University.

Chromatographic system and conditions
A liquid chromatographic-electrospray ionization-mass spectrometry (LC-ESI-MS) system was employed. A Shimadzu LCMS-2010 EV liquid chromatograph mass spectrometer system (Kyoto, Japan) connected to the LC portion consisting of two LC-10AD pumps, a SIL-10AD VP auto injector, a SPD-10A VP UV detector, and a SCL-10A VP system controller was used. Data analysis was accomplished using Shimadzu LCMS Solutions Version 3 software. The mass spectrometer conditions consisted of a curved desolvation line (CDL) temperature of 200˚C and a block temperature of 200˚C. The detector voltage was 1.5 kV. Vacuum was maintained by an Edwards ® E2M30 rotary vacuum pump (Edwards, UK). Liquid nitrogen (Washington State University Central Stores) was used as a source of nebulizer gas (1.5 L/min). A Phenomenex ® Luna ® C 18 (2) column (250 x 4.60 mm, 5 µm) was utilized. The mobile phase consisted of acetonitrile, water, and formic acid (30:70:0.1, v/v/v) that was filtered (0.2 µm ) and degassed under reduced pressure prior to use. Separation was carried out isocratically at a flow rate of 0.6 ml/min. Vorinostat and the internal standard, daidzein ( Figure 1B) were monitored in selected ion monitoring (SIM) positive mode at m/z 265 and 255, respectively.

Stock and working standard solutions
Methanolic stock solutions of vorinostat (100 µg/ml) and the internal standard, daidzein (100 µg/ml) were prepared, protected from light, and stored at -20˚C between uses for no longer than 3 months. Calibration standards were prepared daily from the stock solution of vorinostat by sequential dilution with either rat serum or urine as the matrix, forming a series of concentrations. For serum, the concentrations used were 0.05, 0.5, 1, 5, 10 and 50 µg/ml with three quality control concentrations namely, 0.1, 12.5 and 25 µg/ml. For urine, the concentrations used were 0.5, 1, 5, 10, 12.5 and 25 µg/ml with quality control concentrations at 0.75, 7.5 and 18.75 µg/ml.

Sample preparation
To the working standards or samples (100 µL), 50 µL of internal standard solution (100 µg/ml) was added. One ml ice-cold acetonitrile was added to serum samples for efficient precipitation of plasma proteins. Urine samples were extracted with 0.5 ml of methanol. Samples were then vortexed for 30 s (Vortex Genie-2, VWR Scientific, West Chester, PA, USA) and centrifuged at 10,000 rpm for 5 min at room temperature (25 ± 1˚C) (Beckman Microfuge centrifuge, Beckman Coulter Inc., Fullerton, CA, USA). The supernatant was transferred to new eppendorf tubes and evaporated to dryness by a stream of nitrogen gas. The residue was reconstituted with 100 µL of mobile phase, vortexed for 30 s, and centrifuged at 10,000 rpm for 5 min at room temperature (25 ± 1˚C). The supernatant was transferred to HPLC vials and 10 µL was injected into the LC/MS system.

Precision, accuracy, and recovery
The within-run precision and accuracy of six replicate assays were examined using six different concentrations of vorinostat over the concentration range 0.05-50 µg/ml in serum and 0.5-25 µg/ml in urine used to construct calibration curves in both matrices. As well, the within-run precision and accuracy were investigated for the three quality control (QC) samples, namely 0.1, 5, and 25 µg/ml in rat serum and 0.75, 7.5 and 18.75 µg/ml in rat urine. The between-run precision and accuracy of the assays were determined from the results of six replicate assays on six different days during a one-week period for both calibration and QC samples. The precision was evaluated by the relative standard deviation (RSD). The accuracy was estimated based on the mean percentage error of measured concentration to the actual concentration. Recovery (extraction efficiency) of vorinostat from rat serum and urine was assessed using three concentrations namely 0.05, 5.0, and 50 .0 µg/ml in rat serum and 0.5, 5.0 and 25 in rat urine (n = 3) by comparing the peak area ratio (PAR) of sample normally prepared in the respective matrix as described above to the PAR of the corresponding reference sample prepared by adding a working standard solution to a processed blank sample.

Stability of vorinostat samples
The freeze-thaw stability of vorinostat was tested using three concentrations representing low, middle and high concentrations over the used range including the three QC samples, namely 0.05, 5.0, and 50 .0 µg/ml in rat serum and 0.5, 5.0 and 25 in rat urine. A freshly prepared stock solution of vorinostat (100 µg/ml) in methanol was used for the samples. These samples were analyzed without being initially frozen and then stored at -20 ˚C and thawed at room temperature (25 ± 1˚C) for three cycles.
The stability of vorinostat in normally prepared serum and urine samples during run-time in the LC/MS auto-injector was investigated (n = 3) using samples of three concentration levels, above-mentioned for both matrices under freeze-thaw stability. Samples were prepared and injected directly after preparation, then kept in the sample rack of the auto-injector and re-injected into the LC/MS system 24 h after preparation. Finally, the PAR values of the latter were compared to the former.

Pharmacokinetic disposition of vorinostat in rats
Male Sprague-Dawley rats (234-275 g) were obtained from Simonsen Labs (Gilroy, CA, USA) and given food (Purina Rat Chow 5001) and water ad libitum in our animal facility for at least 3 days before use. Rats were housed in temperature-controlled rooms with a 12 h light/dark cycle. The day before the pharmacokinetic experiment, the right jugular veins of the rats were catheterized with sterile silastic cannula (Dow Corning, Midland, MI, USA) under isoflurane anesthesia (Isothesia ® , Butler Animal Health Supply, Dublin, OH, USA). This involved exposure of the vessel prior to cannula insertion. After cannulation, the Intramedic PE-50 polyethylene tubing (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) connected to the cannula was exteriorized through the dorsal skin. The cannula was flushed with 0.9% saline. The animals were transferred to metabolic cages and fasted overnight. Animal use protocols were approved by The Institutional Animal Care and Use Committee at Washington State University, in accordance with "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985).

Data analysis of rat serum and urine
Quantification was based on calibration curves obtained using the PAR of vorinostat to the internal standard, against vorinostat concentrations using unweighted least squares linear regression. Pharmacokinetic analysis was completed using data from individual rats for which the mean and standard error of the mean (SEM) were calculated for each group except for T max which was represented as median and range. Pharmacokinetic parameters were estimated using WinNonlin ® using noncompartmental analysis. The apparent terminal elimination rate constant (K E ) was estimated from the slope of the loglinear phase of declining serum concentration vs. time plot. The halflife (t 1/2 ) was calculated using the following equation: t 1/2 = 0.693/K E . The area under the concentration time curve (AUC 0-Clast ) was calculated using the linear/logarithmic trapezoidal method. Summation of (AUC 0-Clast ) and the concentration at the last measured point divided by K E yielded AUC 0-∞ . Mean residence time (MRT) was calculated by dividing AUMC 0-∞ by AUC 0-∞ , and clearance (CL) by dividing dose by AUC 0-∞ . The fraction excreted in urine unchanged (F e ) was calculated by dividing the total cumulative amount of vorinostat excreted in urine (∑Xu) by the dose, renal clearance (CLrenal) by multiplying F e by CL, and non-renal clearance (CL NR ) by subtracting CL renal from CL. The C max and corresponding T max were obtained directly from the observed data. Absolute bioavailability (F) was determined by dividing AUC 0-∞, oral by AUC 0-∞ , IV with dose adjustment.

Results and Discussion
Chromatography Separation of vorinostat and daidzein (IS) in rat serum and urine was successfully achieved. No interfering peaks were co-eluted with the investigated compounds ( Figure 2). The retention times of vorinostat and daidzein were approximately 10.5 and 17 min, respectively.
The performance of the LC/MS assay was evaluated according to the following parameters: peak shape and purity, interference from endogenous substances in serum or urine, linearity, limit of quantitation (LOQ), freeze-thaw stability, stability of reconstituted extracts, precision and accuracy. Various compositions of the mobile phase were tried for the best resolution of the drug and internal standard. The optimal separation was obtained with the use of a mobile phase consisting of acetonitrile, water and formic acid (30:70:0.1v/v/v) and a flow rate 0.6 ml/min. Acceptance criteria were assessed in accordance with US FDA bioanalytical method validation guidelines [17].

Linearity and limit of quantitation (LOQ)
Excellent linear relationships were found between the PAR of vorinostat to the internal standard and the corresponding concentrations of vorinostat in both serum (r 2 ≥0.999) over a range of 0.05-50 µg/ml and urine (r 2 ≥0.998) over a range of 0.5-25 µg/ml. The mean regression lines from the validation runs were described by vorinostat (µg/ml) = 0.0280 ± 0.00015x + 0.0032 ± 0.00020 for serum and = 0.0310 ± 0.000067x + 0.0036 ± 0.000046 for urine. The lower LOQ of this assay was 0.05 µg/ml in rat serum and 0.5 µg/ml in rat urine with the respective between-day relative standard deviation of 12.91% and 5.41%; and respective between-day bias of -8.31% and -14.35% for vorinostat. The upper LOQ of this assay was 50 µg/ml in rat serum and 25 µg/ml in rat urine with the respective between-day relative standard deviation of 0.13% and 0.78%; and respective between-day bias of 0.09% and -0.47% for vorinostat. The back-calculated concentrations were within the acceptance criteria.

Precision, accuracy and recovery
The within-and between-run precision (RSD) calculated during replicate assays (n = 6) of vorinostat in rat serum and urine was <15% over a wide range of concentrations ( Table 1). The intra-and inter-run bias determined during the replicate assays for vorinostat was within the acceptable range: -15% to 15% (Table 1). The recovery (extraction efficiency) of vorinostat was assessed over the three selected samples representing the low, middle and high concentrations of the working range in both serum (0.05, 5, 50 µg/ml, n = 3) and urine (0.5, 5, 25 µg/ ml, n = 3). The recovery values were ≥ 99.83% in serum and ≥ 97.45% in urine, (Table 2). These data indicate that the developed LC/MS method is reproducible and accurate.

Stability of vorinostat samples
The stability of vorinostat following three freeze-thaw cycles and storage in the auto-injector at room temperature (25 ± 1˚C) for 24 h are reported in (Table 2). The percentage recovery of vorinostat was ≥ 97.56% in rat serum and ≥ 97.48 in rat urine following three freezethaw cycles indicating no degradation of this compound in either  Table 2: Recovery and stability of vorinostat in rat serum and urine (n = 3, mean, and SD).
matrix. The percentage recovery of vorinostat was ≥ 98.74% in serum and ≥ 99.05% in urine after 24 h storage in the auto-injector at room temperature (25 ± 1˚C) confirming the stability of this compound in both matrices under these conditions.

Pharmacokinetics of vorinostat in rats
The LC/MS method was successfully applied to the determination of vorinostat in a pre-clinical pharmacokinetic study in male Sprague-Dawley rats (n = 3 for each treatment group). Following administration of vorinostat either intravenously (10 mg/kg) or orally (50 mg/ kg), the disposition in serum was investigated (Figure 3). The serum concentration versus time profiles observed for vorinostat in PEG400 shows that vorinostat was rapidly cleared from the body and was detected only up to 2 h post-dosing, ( Figure 3A). Vorinostat in PEG400 after intravenous administration demonstrated a short half-life (t 1/2 = 0.57 ± 0.05 h), low area under curve (AUC 0-∞ = 2.46 ± 1.61 µg.h/ml), short mean residence time (MRT = 0.54 ± 0.13 h) and a rapid serum clearance (CL = 8.93 ± 4.02 L/h/kg). Similar results of vorinostat in rat serum have been reported following intravenous administration at a dose of 2 mg/kg [14].
Following oral administration at 50 mg/kg, vorinostat in 2% methylcellulose (MC) suspension was detected in serum up to 12 h, ( Figure 3B). The serum concentrations of vorinostat given orally appeared to decline with a mean serum elimination half-life (t 1/2 ) of 3.22 ± 0.55 h. The area under the curve, representing the total amount of drug exposure in the serum over time, was 0.99 ± 0.12 h.µg/ml. The absorption was fast as indicated by a short time to maximum concentration (T max = 0.25 as median with a range of 0.75) and a concentration maximum (C max ) of 0.21 ± 0.03 µg/ml. However, the absolute bioavailability was low (7.03 ± 0.63%). Previous pharmacokinetic studies also report a short T max (0.17 ± 0.0 h) and low oral bioavailability (6.9%) of vorinostat following oral administration of (5 mg/kg) to Wistar rats [13]. In another study, such short T max (0.3 ± 0.1 h) and low oral bioavailability (11%) of vorinostat following oral (10 mg/kg) dosing of rats were also documented [14]. The authors suggested this was related to a high firstpass effect and predominant hepatic biotransformation. Herein, the total oral clearance of vorinostat (CL/F) was determined to be 52.09 ± 6.30 L/h/kg, which was predominated by non-renal clearance (CL NR /F = 51.80 ± 6.28 L/h/kg) with only minor renal clearance (CL renal /F= 0.29 ± 0.03 L/h/kg).
Following intravenous administration, the total amount excreted in urine plot (Figure 4) illustrates that only a fraction of the vorinostat dose was excreted in the urine following both intravenous and oral administration. This small amount of vorinostat was excreted in urine by 72 h following intravenous administration and 24 h following oral administration ( Figure 4A, 4B, respectively). The fraction excreted unchanged (F e ) of vorinostat was 7.54 ± 1.68 % from PEG400 and 0.56 ± 0.02% from 2% MC suspension. Both the amounts of vorinostat excreted unchanged in urine and fraction excreted unchanged (F e ) following oral administration were lower than those experienced after intravenous dosing indicating that the hepatic metabolism is a major contributor of vorinostat elimination. This agrees with the previously reported findings of predominant hepatic biotransformation of vorinostat [14].

Conclusion
The developed LC/MS method for vorinostat is sensitive, reproducible, specific, linear over a suitable concentration range, precise and accurate. No degradation of vorinostat in rat serum and urine was detected following three freeze-thaw cycles and storage in the auto-injector at room temperature for 24 h. In addition, this assay method has been applied successfully in a pre-clinical study of the pharmacokinetics of vorinostat in rat serum and urine after intravenous and oral administration and the results were in agreement with previously reported data. Using this method, it is possible to characterize and evaluate newly developed drug delivery systems of vorinostat intended for oral and intravenous administration with improved serum and urine disposition.


text:
Synthesis, Spectroscopic Characterization, and In Vitro Antimicrobial Studies of Pyridine-2-Carboxylic Acid N′-(4-Chloro-Benzoyl)-Hydrazide and Its Co(II), Ni(II), and Cu(II) Complexes

N-substituted pyridine hydrazide (pyridine-2-carbonyl chloride and 4-chloro-benzoic acid hydrazide) undergoes hydrazide formation of the iminic carbon nitrogen double bond through its reaction with cobalt(II), nickel(II), and copper(II) metal salts in ethanol which are reported and characterized based on elemental analyses, IR, solid reflectance, magnetic moment, molar conductance, and thermal analysis (TG). From the elemental analyses data, 1 : 2 metal complexes are formed having the general formulae [MCl2(HL)2] ·yH2O (where M = Co(II), Ni(II), and Cu(II), y = 1–3). The important infrared (IR) spectral bands corresponding to the active groups in the ligand and the solid complexes under investigation were studied. IR spectra show that ligand is coordinated to the metal ions in a neutral bidentate manner with ON donor sites. The solid complexes have been synthesized and studied by thermogravimetric analysis. All the metal chelates are found to be nonelectrolytes. From the magnetic and solid reflectance spectra, the complexes (cobalt(II), nickel(II), and copper(II)) have octahedral and square planner geometry, respectively. The antibacterial and antifungal activity's data show that the metal complexes have a promising biological activity comparable with the parent ligand against bacterial and fungal species.

Introduction
Hydrazones and their metal complexes have been given remarkable attention by the researchers, since they act as the most important stereochemical models in transition metal coordination chemistry, due to their preparative accessibility and structural variety. It has been suggested that the carbonyl linkage (C=O) in Hydrazide is responsible for their biological activities such as antitumor, antibacterial, antifungal, and herbicidal activities [1,2]. Recently, the developments of new metal drug complexes have received special interests in the field of coordination chemistry [3,4]. The transition metal-coordinated Hydrazones complexes play a significant role in many catalytic reactions like oxidations, asymmetric cyclopropanation, and polymerisation [5][6][7]. Hydrazide complexes of transition metal ions are known to provide useful models for elucidation of the mechanisms of enzyme inhibition by hydrazine derivatives [8] and for their possible pharmacological applications [9]. The activities of some such type complexes are very significant against Gram-positive bacteria in vitro. The derivatives of these chelate act as good potential oral drugs to treat the genetic disorders like thalassemia [10]. The structural characterization of these resultant hydrazide complexes revealed some interesting facts, such as their tendency and potency to act as planar pentadentate ligands in most of the complexes [11][12][13][14] along with tridentate character [15]. Moreover, these ligands exhibit ketoenol tautomerism and can coordinate in neutral [16], monoanionic [17], dianionic [4,15,16], or tetraanionic [18] forms, to the metal ions which have coordination numbers of six and seven [4,18], generating mononuclear or binuclear species. However, it depends on the reaction  conditions, such as metal ion, its concentration, the pH of the medium, and the nature of the hydrazide ligand [19]. Certain hydrazides and their Cu(II) complexes have antitumor activity. Transition metal complexes having the ability to bind and cleave DNA under physiological conditions are of current interests for their potential applications [20] in nucleic acids chemistry. Such complexes [21] are useful in foot printing studies, as sequence specific DNA binding agents, in genomic research and as diagnostic agents in medicinal applications. Much attention has been paid to biologically active metal complexes in recent years. Oxygen and nitrogen donor ligands [22] have been widely studied due to their high potential to coordinate with transition metals. Compounds containing carbonyl oxygen groups [23] have important position among organic reagents as potential donor ligands for the transition metal ions. Organic compounds and metal complexes [24] both display a wide range of pharmacological activity including anticancer, antibacterial, and fungi static effects. Though some reports are available on the hydrazones metal complexes [21,22], there is no work focussed in the hydrazide derived from pyridine-2-carboxylic acid N -(4-chloro-benzoyl)-hydrazide (HL) and its complexes (Scheme 1). In this paper, we report the synthesis, spectroscopic, thermal, and biological studies of the newly synthesized hydrazone complexes of the transition metal ions (Co(II), Ni(II), and Cu(II)).

Biological
Activity. Antimicrobial activity of the tested samples was determined using a modified Kirby-Bauer disc diffusion method [25]. One hundred microliters of the tested bacteria or fungi were grown in 10 mL of fresh media until they reached account of approximately 98 cells/mL for bacteria and 95 cells/mL for fungi [26]. One hundred microliters of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Isolated colonies of each organism that might be playing a pathogenic role should be selected from primary agar plates and tested for susceptibility by disc diffusion method [27]. Of the many media available, NCCLS recommends Mueller-Hinton agar due to its results in good batch-to-batch reproducibility. Disc diffusion method for filamentous fungi tested by using approved standard method (M38-A) developed [28]. For evaluating the susceptibilities of filamentous fungi to antifungal agent, the disc diffusion method was applied for yeast developed by using approved standard method (M44-P) [29].  [29]. Standard discs of Gentamycin (antibacterial agent) and amphotericin-B (antifungal agent) served as positive controls for antimicrobial activity, but filter discs impregnated with 10 ML of solvent (distilled water, chloroform, DMSO) were used as a negative control. The agar used is Mueller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented, and standard zones of inhibition have been determined for susceptible and resistant values. Blank paper discs (Schleicher and Schuell, Spain) with a diameter of 8.0 mm were impregnated with 10 ML of tested concentration of the stock solutions. When a filter paper disc impregnated with a tested chemical is placed on agar, the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar, it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as zone of inhibition or clear zone. For the disc diffusion, the zone diameters were measured with slipping calipers of the national committee for clinical laboratory standards [29]. Agar-based methods such Etest and disc diffusion can be good alternatives because they are simpler and faster than broth-based methods [30].

Result and Discussion
The were the evidence of the secondary amide bonding to the ligand [34]. 13 C NMR spectrum of complexes displays a signal at δ 167.45 ppm and δ 163.54 ppm, due to (C-NH) and (C=O), which was indicating that carbon atoms of carbon-amide group and oxygen atom of carbonyl group participate the formation of ligand [28]. The spectrum shows the molecular ion peak at m/z = 275 (C 13 H 10 ClN 3 O 2 , calculated atomic mass 276 amu due to 13 [29]. The electronic spectra of the cobalt(II) complex showed three bands at 8780-8810, 17475-17775 and 30235-30270 cm −1 , which may be assigned to 4 T 1g → 4 T 2g (F), 4 T 1g → 4 T 1g (P), and 4 T 1g → 3 A 2g (F) transitions and suggested octahedral geometry around the cobalt ion. The magnetic moment 4.80-488 BM is an addition evidence for an octahedral structure [35]. The cobalt complexes showed magnetic moment values 4.70-490 B.M. at room temperature. These high values of the magnetic moments and the stoichiometries suggest a coordination number of six for the central cobalt ions and an octahedral geometry. The magnetic properties of copper complex may be divided into broad classes. First, those are having essentially temperature-independent magnetic moments in rang 2.20 BM. Those exhibiting such moments are mononuclear complexes having no major interaction between the unpaired electrons on different copper ion. The moments in such complex, as in apparent, lie appreciably above the spine-only value (1.73 BM), but as the electronic ground states are nondegenerate, this cannot arise from inherent angular momentum in the ground state. It arises due to mixing of some orbital angular momentum from excited states via spin orbit coupling. The copper (II) complexes exhibit magnetic moments of 1.70-1.75 B.M.,  respectively, at room temperature. The electronic spectra of the copper(II) complexes display a broad band at 14220-14918 cm −1 due to 2 B 1g → 2 E g and two bands at 16390-16550 and 27250-27350 cm −1 assigned to d-d transitions and a charge transfer band, respectively, of square planner environment [31]. The observed magnetic moment of the copper complexes is 1.75-180 BM. The Ni(II) complex is found to have a room temperature magnetic moment value of 3.87 B.M., which is in the normal range observed for octahedral Ni(II) complexes [26,34,36]. The electronic spectrum displays three bands in the solid reflectance spectrum at m 1 : 14968 cm −1 ; 3 A 2g → 3 T 2g ; m 2 : 17,788 cm −1 ; 3 A 2g → 3 T 1 g (F) and m 3 ; 21347 cm −1 ; 3 A 2g → 3 T 1g (P). The spectrum shows also a band at 24565 cm −1 which may be attributed to ligand to metal charge transfer (Figure 1). The maximum conductivity value was 1.81 × 10 −4 ohm −1 cm −1 for doped copper(II) complex because of the small size of nickel atom compared with iron atom. Also the prepared hydrazide complexes at different temperatures (303-373) K show that the increased conductivity with increasing of temperatures may be attributed to presence of metals (dd * ) transition. Figures 2 and 3 show that the conductivities of doped and undoped compounds increase with increasing of temperatures which is consistent with semiconductors properties [19,20].
The dopping compounds have higher conductivities than undoped because iodine doping leads to oxidation of iodine molecules to form I 3 −1 , I 5 −3 , and reduction of hydrazide complexes molecules, this effect increase the conductivity by making acceptor bands, and the distance between the energy levels was low. The DC electrical conductivities of doped and undoped prepared compounds over temperature range (0-110 • C) and under vacuum were measured by using conductivity apparatus which is consisting of temperature recorder, power supply, voltmeter, resistance and sample cell. The studied samples were as discs covered in two sides by silver paint. The ligand, Cu(II), and Ni(II) complexes were doped with iodine by mixing 1 g (0.004 mole) ligand, 1 g (0.0022 mole) Cu-complex, and 1 g (0.002 mole)  Ni-complex with 25 mL of iodine solution in CCL 4 (4%, w/v), the mixture was refluxed with stirring for 48 hours and then filtered and dried in the vacuum oven at 50 • C. The conductivities at different temperatures were calculated according to Arrhenius equation as shown below [19]: The resistance of the sample and its electrical conductivity is calculated from the equations: where R s is standard resistance (ohm), R x is sample resistance (ohm), V s is standard Voltage (volt),V x is sample voltage (volt), L is Sample thickness (cm), and A is The painted area of the sample surface (cm 2 ).

Biological Activity.
In testing the antibacterial activity of these compounds we used more than one test organism to increase the chance of detecting antibiotic principles in tested materials. The sensitivity of a microorganism to antibiotics and other antimicrobial agents was determined by the assay plates which incubated at 28 • C for 2 days for yeasts and at 37 • C for 1 day for bacteria. All of the tested compounds showed a remarkable biological activity against different types of Gram-positive and Gram-negative bacteria and against fungi species. The data are listed in Table 1.
It was demonstrated that the newly prepared ligand and its metal complexes showed a higher effect on C. albican (Gram-negative bacteria) and S. aureus (Gram-positive bacteria). It is known that the membrane of Gram-negative bacteria is surrounded by an outer membrane containing lipopolysaccharides. The newly synthesized hydrazide and its metal complexes seem to be able to combine with the lipophilic layer in order to enhance the membrane permeability of the Gram-negative bacteria. The lipid membrane surrounding the cell favours the passage of only lipid soluble materials; thus the lipophilicity is an important factor that controls the antimicrobial activity. Also the increase in lipophilicity enhances the penetration of hydrazide and its metal complexes into the lipid membranes and thus restricts further growth of the organism. This could be explained by the charge transfer interaction between the studied molecules and the lipopolysaccharide molecules which lead to the loss of permeability barrier activity of the membrane. The hydrazide and its metal complexes could enhance the antimicrobial effect on both strains probably by the hydroxyl group. The Co(II), Ni(II), and Cu(II) complexes were almost the most promising broad spectrum antimicrobial agents due to the presence of coordinated anion with higher antimicrobial activity than the other complexes. From the data the inhibition zone of the metal chelates is higher than that of the ligand. Such increased activity of the metal chelates is due to the lipophilic nature of the metal ion in complexes. Furthermore, the mode of action of hydrazide of the compounds may involve the formation of a hydrogen bond through the azomethine nitrogen atom with the active canters of all the constituents, resulting in interference with normal cell process.

Conclusion
The results of this investigation support the suggested structures of the metal complexes. It is obvious from this study that only mononuclear complexes are obtained. The IR spectral studies reveal that HL coordinated to the metal ions via C=O and amide NH-NH group. The chelates are nonelectrolytes. All metal cations have octahedral geometry except copper (II) chelate. The thermal decomposition of the complexes as well as the thermodynamic parameters is studied. The biological activities of the hydrazide under investigation and its complexes against bacterial and fungal organisms are promising which need further and deep studies on animals and humans.


extfieldsofstudy: Medicine Chemistry

text:
Binaphthyl-anchored antibacterial tripeptide derivatives with hydrophobic C-terminal amino acid variations

The facile synthesis of seven new dicationic tripeptide benzyl ester derivatives, with hydrophobic group variations in the C-terminal amino acid component, is described. Moderate to good activity was seen against Gram-positive bacteria in vitro. One cyclohexyl-substituted compound 2c was tested more widely and showed good potency (MIC values ranging from 2–4 μg/mL) against antibiotic-resistant strains of Staphylococcus aureus and Enterococci (VRE, VSE), and against Staphylococcus epidermidis.

Introduction
Among the most pressing challenges in current healthcare is the resistance of bacterial human pathogenic organisms to antibiotics [1,2], and of particular concern is the resistance to the cationic glycopeptide, vancomycin [3,4]. This challenge is being addressed in a number of ways, which include both detailed studies aimed at the further understanding of the mechanism of this resistance, as well as the development of new large glycopeptide analogues containing amine sites that can be protonated, such as telavancin, oritavancin and dalbavancin [5]. An alternative approach to meeting this resistance challenge, at least in part, is through the design and synthesis of smaller cationic peptidic compounds incorporating features that could circumvent the vancomycin resistance mechanism. In much of our work we have been concerned with binaphthyl-based dicationic peptide derivatives, for example the acyclic tripeptides of type 1 (Figure 1; for example A = CH 2 CH=CH 2 ,  [6]), which show significant promise as antibacterials [6][7][8]. In the development of these antibacterials, it became apparent that the nature of the C-terminal amino acid derivative was significant, with indications that the hydrophobic groups at the terminus (Figure 1, group B) [6] and at the α-carbon of the amino acid moiety (Figure 1, group A) [6,7] were important. In further exploration of the structural space in the latter area, while maintaining benzyl ester functionality at the terminus itself (a free carboxylic acid unit at the C-terminal was deleterious to activity [8]), we envisaged the introduction of two alkyl substituent units incorporated in a ring system at this carbon, resulting in the target compounds 2a-d. Two further compounds based on a β-alanine unit with disubstitution at the α-or β-carbons of this unit, 2f and 2e respectively, were accessed with a view to assessing the effect of variation in the spatial disposition of the cycloalkyl or oxacycloalkyl ring on antibacterial activity. The conformationally less restricted gem diethylsubstituted compound 2g was also targeted in order to make antibacterial activity comparisons with the five-membered ring analogue 2b. The results of these synthetic and antibacterial testing studies are reported in this paper.

Results and Discussion
A concise and flexible approach to all the target compounds 2a-g was used, starting from the commercially available amino acid derivatives 3 and proceeding via the amino benzyl esters 4 (Scheme 1), with the exception of the synthesis of 2c in which the commercially available 4c was used as the starting material. Addition of the protected central arginine unit by diimide-or BOP-induced amide bond formation, followed by selective Fmoc removal from the respective intermediates 5, then provided access to the key intermediate amines 6a-g (Scheme 1). Diimide-mediated coupling of the previously reported lysine containing (S)-binaphthyl acid derivative 7 [8] then afforded the protected tripeptides 8a-g. Removal of the Pmc (or Pbf) and Boc protecting groups in one pot was then achieved by exposure to trifluoroacetic acid, followed by trifluoroacetate/chloride ion exchange on treatment with an excess of HCl in diethyl ether, and finally evaporation to afford the salts 2a-g in good overall yields.
The structures of the final compounds were supported by 1 H and 13 C NMR spectroscopy and high-resolution mass spectrometry. Details of the synthesis and spectral data are given in the experimental section for the representative compound 2c. The experimental and spectroscopic data for all the other compounds are included in Supporting Information File 1.
The peptidic dihydrochloride salts 2a-g were tested against the Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and four clinical isolates of vancomycin-resistant (and sensitive) enterococci (VRE; Enterococcus faecium), and the results are shown in Table 1; some compounds were also tested against Staphylococcus epidermidis (ATCC 12228). Vancomycin was used as a positive control, and showed a rounded MIC (minimum inhibitory concentration) value of 2-3 μg/mL against S. aureus and MIC values of 2, >25, >25 and 3 μg/mL against the vancomycin sensitive and partially resistant enterococci strains, VRE 243 , VRE 449 , VRE 820 and VRE 987 , respectively (Table 1). For comparison purposes, data for the previously reported [6] isobutyl-substituted analogue 9 (Table 2) are also included.
While MIC micromolar values should be used for comparative analysis of activities, the more commonly used concentration of micrograms per milliliter is retained in this case. The micromolar values are barely different from the latter, as the molecular weights for all of the compounds are similar (959-1001; vancomycin·HCl, 1486). In most cases, incorporation of a hydrophobic alkyl ring in the chain adjacent to the ester func-tionality results in good activity against S. aureus and S. epidermidis, but a greater variation of activity was seen with the enterococcal strains. The systems with four-and fivemembered cycloalkyl rings (2a and 2b) displayed similar activities, while the cyclohexyl analogue 2c was somewhat better.
Reducing the hydrophobicity while increasing the hydrophilicity of the six-membered ring in 2c through inclusion of a ring oxygen atom, as in 2d, had a significant detrimental effect on the activity against the vancomycin-resistant strains VRE 449 and VRE 820 . Interestingly, the placement of an extra methylene group in the chain, either on the carboxylic ester side (2e) or the amino side (2f), had no or little effect on antibacterial activity, regardless of whether a ring oxygen atom was present or not. It appeared that moving the hydrophobic ring substituent position by a small amount in the terminal amino acid unit could be tolerated in these systems.
The diethyl-substituted peptide derivative 2g was more active than the constrained ring comparator compound 2b against the enterococci, including the vancomycin resistant isolates VRE 449 and VRE 820 , but both had similarly good potencies against S. aureus. In contrast, compound 2c with the cyclohexyl ring was somewhat more active than the isobutyl-substituted analogue 9. It would thus appear that greater coverage of the hydrophobic space available in this region is required for good to moderate activity against both the staphylococci and enterococci.
As a result of its promising initial activity profile, the tripeptide derivative 2c was subjected to further evaluation ( showed good activity against all these Gram-positive bacteria with MIC values in the range of 2-4 µg/mL. Again, for comparison purposes, results for the published analogue 9 [6], containing a single isobutyl group in place of the cyclohexyl group, are also included in Table 2. This tripeptide derivative 9 showed similar, if slightly weaker, antibacterial activity against these strains, apart from VSE, against which it was significantly less potent than 2c. Although the mode of action of the tripeptide derivatives has not been established, our earlier results on compounds of type 1 ( Figure 1) and related cyclic systems implicated the possibility of more than one mode of action [8]. As noted for other cationic peptide derivatives [9][10][11][12], cell membrane damage could well be one of these actions, together with some more specific interactions. Dual-action type behaviour has been shown for the vancomycin analogue telavancin, which affects cell wall synthesis and the integrity of the cell membrane [13].

Conclusion
In conclusion, seven novel binaphthyl-anchored tripeptide derivatives have been prepared and tested for antibacterial activity against S. aureus and four clinical enterococcal strains. The dicationic derivatives 2c and 2g showed the best overall activities. In addition, compound 2c with a hydrophobic cyclohexyl substituent originating from the α-position of the C-terminal amino acid ester, showed good activity against S. epidermidis and MSSA, MRSA, VISA and VRE organisms. Our results confirmed the positive contribution to good Grampositive antibacterial activity, engendered by filling of a hydrophobic area close to the C-terminus of these acyclic tripeptide derivatives.

Experimental
General notes were those detailed previously in the supporting information for reference [8].

Supporting Information
Supporting Information File 1 Experimental procedures and associated spectroscopic data (NMR and MS) for the syntheses of compounds 2a-b and 2d-g.


text:
2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl butyrate

The title compound, C18H26O11, was synthesized by a condensation reaction of 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide and butyric acid. The acetoxymethyl and butyrate groups are located on the same side of the pyran ring, showing the β configuration for the d-glycosyl ester; the butyl group adopts an extend conformation, the C—C—C—C torsion angle being 179.1 (7)°. In the crystal, the molecules are linked by weak C—H⋯O hydrogen bonds.

The title compound, C 18 H 26 O 11 , was synthesized by a condensation reaction of 2,3,4,6-tetra-O-acetyl--d-galactopyranosyl bromide and butyric acid. The acetoxymethyl and butyrate groups are located on the same side of the pyran ring, showing the configuration for the d-glycosyl ester; the butyl group adopts an extend conformation, the C-C-C-C torsion angle being 179.1 (7) . In the crystal, the molecules are linked by weak C-HÁ Á ÁO hydrogen bonds.

Comment
Carbohydrates provide excellent platforms upon which to explore unique features for the drug-discovery process. Numerous natural glycosyl esters such as phyllanthostatin family (Li et al., 1992) and dimeric ellagitannin coriariin A have been total synthetized (Smith et al., 1986). Some of them were proved to possess anti-tumor activities (Feldman et al., 2000). Also the glycosyl esters have long drawn attention as potential glycosyl donors. Several crystal structures of carbohydrate derivatives were reported (Sambaiah et al., 2001;Parkanyi et al., 1987;Roslund et al., 2004;Liu et al., 2009;Kumar et al., 2005).
Recently we have synthetized the title compound and report its crystal structure herein.
The molecular structure of the title compound is shown in Fig. 1. In the molecule, the acetoxymethyl and butyrate groups are located on the same side of the pyran ring, showing the β-configuration for the D-glycosyl ester; the butyl group adopts an extend conformation, the C6-C7-C8-C9 torsion angle being 179.1 (7)°. The molecules are linked by weak C-H···O hydrogen bonding in the crystal.

Refinement
Methyl H atoms were placed in calculated position with C-H = 0.96 Å and torsion angle was refined from electron density with U iso (H) = 1.5U eq (C). Other H atoms were placed in calculated positions with C-H = 0.97-0.98 Å, and included in the final cycles of refinement in riding model with U iso (H) = 1.2U eq (C). As no significant anomalous scatterings, Friedel pairs were merged. The enantiomer has been assigned by reference to the unchanging chiral C5 atom in the synthetic procedure.


extfieldsofstudy: Biology Medicine

text:
Metal Ion Chelates as Surrogates of Nucleobases for the Recognition of Nucleic Acid Sequences: The Pd2+ Complex of 2,6-Bis(3,5-dimethylpyrazol-1-yl)purine Riboside

A 2,6-bis(3,5-dimethylpyrazol-1-yl)purine ribonucleoside has been prepared and incorporated as a conventionally protected phosphoramidite into a 9-mer 2′-O-methyl oligoribonucleotide. According to 1H NMR spectroscopic studies, this nucleoside forms with Pd2+ and uridine a ternary complex that is stable at a micromolar concentration range. CD spectroscopic studies on oligonucleotide hybridization, in turn, suggest that the Pd2+ chelate of this artificial nucleoside, when incorporated in a 2′-O-methyl-RNA oligomer, is able to recognize thymine within an otherwise complementary DNA strand. The duplex containing thymidine opposite to the artificial nucleoside turned out to be somewhat more resistant to heating than its counterpart containing 2′-deoxycytidine in place of thymidine, but only in the presence of Pd2+. According to UV-melting measurements, replacement of 2′-O-methyladenosine with the artificial nucleoside markedly enhances hybridization with a DNA target, irrespective of the identity of the opposite base and the presence of Pd2+. With the thymidine containing DNA target, the T m value is 2–4°C higher than with targets containing any other nucleoside opposite to the artificial nucleoside, but the dependence on Pd2+ is much less clear than in the case of the CD studies.

Introduction
In nature, recognition of nucleic acid sequences is based on Watson-Crick base pairing, while vertical stacking of base pairs accounts for most of the duplex stability. At room temperature, 10 base pairs are typically required for a stable duplex, meaning that targeting sequences shorter than this by the conventional strategy, that is, using complementary oligonucleotides, is problematic. Sequences within a double-stranded region are also difficult to target because thermodynamics favor formation of the longer duplex. The first problem has partly been addressed by using small molecules exhibiting high affinity towards certain specific short sequences [1]. No clear-cut relationship exists between the target sequence and the structure of the small molecule, however, making the design and preparation of such molecules a demanding task. PNA oligonucleotides offer a solution to the second problem, as PNA/DNA heteroduplexes are more stable than native double-stranded DNA, but even in the best cases a substantial excess of the PNA is still needed for efficient invasion [2,3].
Oligonucleotides composed of unnatural monomers with an enhanced affinity towards their complement nucleobases could potentially form stable duplexes even with short target sequences as well as invade double-stranded DNA even when present in only stoichiometric amounts. One way to achieve the desired high affinity is to exploit the coordination of a ring nitrogen (N1 of purines or N3 of pyrimidines) of the natural nucleobase to a soft metal ion, such as Pd 2+ , carried by an artificial nucleobase [4]. Discrimination between the four natural nucleobases, in turn, could be achieved through a combination of additional destabilizing (steric) and stabilizing (hydrogen bonding) interactions. A number of studies on metal-ion-mediated base pairs have been published [5,6], but the focus has generally been on expanding the genetic code by introducing a completely new artificial base pair [7,8] or impregnating DNA with metal ions for nanotechnological applications [9][10][11], rather than developing high-affinity complements for the natural nucleobases. In the former, both of the ligands of the metalion-mediated base pair may be freely designed for maximum performance (in many cases, the same ligand is used in both strands), whereas in the latter the sole artificial nucleobase must be able to not only coordinate the metal ion but also accommodate the steric and hydrogen bonding requirements of the natural nucleobase, presenting a unique challenge.
In the present study, the Pd 2+ complex of 2,6-bis-(3,5dimethylpyrazol-1-yl)purine ribonucleoside (1) is used as an artificial nucleoside. Nucleoside 1, with no potential for stabilizing hydrogen bond interactions, may be considered as a reference structure for future studies. On a monomeric level, the binding affinity and selectivity of this artificial nucleoside were studied by NMR titrations. For a more realistic model, nucleoside 1 was additionally converted to a phosphoramidite building block (2) and incorporated into a 2 -O-methyl-RNA oligonucleotide. The effect of Pd 2+ on the secondary structure and thermal stability of duplexes between this modified oligonucleotide and complementary 2 -O-methyl-RNA and DNA oligonucleotides was investigated by melting temperature and CD spectrometric studies. For comparison, the same experiments were also performed on a 2 -O-methyl-RNA oligonucleotide having adenosine in the place of the artificial nucleoside.

NMR Spectrometric
Titrations. The formation and stability of the complexes between the artificial nucleoside 1 and uridine in the presence and absence of Pd 2+ were studied by 1 H NMR spectrometric titration. The NMR spectra were recorded in a 120 mM deuterated phosphate buffer at pH 7.3. The initial concentration of the nucleoside components of the putative ternary complex, namely, 1 and uridine, was 3.6 mmol L −1 , while a deficit of Pd 2+ (0.7 eq., 2.5 mmol L −1 as K 2 PdCl 4 ) was employed to ensure exclusive formation of the desired ternary complex. The titration was carried out by diluting the sample with buffer until the uridine concentration was 0.061 mmol L −1 .
Over the entire concentration range studied, two distinct sets of signals were observed for uridine: one referring to the free nucleoside and the other one (presumably) to the ternary complex (Figures 1(a)-1(d)). The two sets of signals were particularly well resolved in the case of H5 and H1 resonance of uridine and the integrals of these signals were used to determine the mole fraction of the complex at each concentration ( Figure 2). To exclude the possibility that the observed changes in the chemical shifts of the H5 and H1 of uridine were caused by Pd 2+ alone, a corresponding experiment with uridine and Pd 2+ but without the artificial nucleoside 1 was carried out (Figures 1(f)-1(i)). In this case, an upfield change in the chemical shifts of the H5 and H1 of uridine was observed, in striking contrast with the downfield shift associated with formation of the putative ternary complex. In other words, the H5 and H1 signals assigned to uridine in its ternary complex with Pd 2+ and the artificial nucleoside 1 were not observed in the absence of the latter. No effort was made to characterize the complexes between uridine and Pd 2+ but the NMR spectra suggest that a number of different relatively weak complexes prevail, again in sharp contrast with the single strong complex formed in the presence of the artificial nucleoside 1.
Upon introduction of Pd 2+ , the signals for the hydrogens on the pyrazole rings of 1 shift downfield by approximately 0.4 ppm. The signals of two of the methyl substituents of the pyrazole rings exhibit an even greater downfield shift (approximately 0.6 and 0.8 ppm, resp.), whereas the other two are shifted slightly upfield. Adding uridine to the system hardly affects the chemical shifts of these protons (data not shown).
As expected, dilution of the sample was accompanied by decrease of the proportion of signals arising from the ternary complex (Figures 1(a)-1(d)). However, even at the lowest concentration employed, that is, 61 μmol L −1 , the mole fraction of the complex was still more than 80% of the saturation level observed at high concentrations. In the absence of Pd 2+ , no complex was formed even though a high concentration (13.84 mmol L −1 ) of the nucleosides was  used ( Figure 1(e)). In all likelihood, Pd 2+ forms a tridentate chelate with the artificial nucleoside 1 by binding to N1 of the purine ring and N2 of the pyrazolyl moieties, the fourth coordination site being filled by the N3 of uridine ( Figure 3) [14]. The ability of thymine to form base pairs mediated by N3-coordinated soft metal ions has previously been demonstrated and uracil most likely exhibits similar behavior [15][16][17][18][19]. While no spectra could be obtained at sufficiently low concentrations to allow for reliable determination of the stability of the complex, the data at hand suggest a dissociation constant in the low micromolar range.

CD Spectropolarimetric Measurements.
To obtain information about the impact of the modified nucleoside and  7X, the situation is quite different: the curves for the "mismatched" duplex 7X:10C in the presence and absence of Pd 2+ overlap with each other and also with the curve for the "full-match" duplex 7X:10T in the absence of Pd 2+ ( Figure 5(b)). In the presence of Pd 2+ , however, duplex 7X:10T is remarkably resistant to heating, losing significantly less of its ellipticity than the other combinations. In other words, the artificial 2,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)purine nucleobase seems to be able to recognize thymine, but only in the presence of Pd 2+ .
With the 2 -O-methyl-RNA homoduplexes between unmodified oligonucleotides, the ellipticity of the matched duplex 7A:8U exhibits a strongly sigmoid curve with an inflection point at approximately 75 • C and the mismatched duplex 7A:8C shows a similar curve with an inflection point at approximately 60 • C (Figure 6(a)). The inflection points are essentially independent on the presence of Pd 2+ , but in the case of the mismatched duplex 7A:8C, the loss of ellipticity at high temperatures is somewhat lower in the presence of Pd 2+ . In contrast, no discrimination between uridine and cytidine is observed with the modified oligonucleotide 7X: both duplexes 7X:8U and 7X:8C exhibit largely similar sigmoid curves with inflection points at approximately 60 • C (Figure 6(b)). In both cases, the maximum loss of ellipticity is somewhat lower in the presence of Pd 2+ . The plots of the duplexes containing multiple mismatches are essentially linear and independent on both the sequence and the presence of Pd 2+ (Figure 7).  Table 2.
As expected, the unmodified oligonucleotide 7A forms the most stable 2 -O-methyl-RNA/DNA heteroduplex with the fully complementary DNA oligonucleotide 10T, but the discrimination is not very strict. For example, the fullmatch duplex 7A:10T exhibits a T m value of only 5 • C higher than the mismatched duplex 7A:10A (50.0 and 45.0 • C, resp.). Adding 1 eq. of Pd 2+ to the oligonucleotide mixtures has little effect on the stability of any of the duplexes studied. Unexpectedly, all of the respective heteroduplexes formed by the modified oligonucleotide 7X turned out to be significantly more stable than the ones formed by 7A, with T m values ranging from 58.6 to 62.6 • C. The most stable duplex was the one between 7X and 10T, although the selectivity was even lower than in the case of 7A. Furthermore, the selectivity is only marginally improved in the presence of Pd 2+ . In

Discussion
The Pd 2+ chelate of the 2,6-bis-(3,5-dimethylpyrazol-1-yl) purine nucleobase presented in this study has no potential for hydrogen bonding interactions with the canonical nucleobases-in other words, its base pairing relies solely on metal ion coordination. On the other hand, the modified nuclobase presents a much greater surface for π-π stacking interactions and the observed Pd 2+   heteroduplexes is probably a result of this enhanced basestacking. The Pd 2+ -dependent selective recognition of a thymine base within a complementary DNA oligonucleotide, in turn, is probably attributable to two factors, namely, the relatively small size of the thymine base, as well as its potential to act as an anionic nitrogen ligand. Of the four native nucleobases, only the pyrimidines may form a base pair of Watson-Crick geometry with the artificial metallo-nucleobase (and even these base pairs would be approximately 1.3Å longer than the natural ones). Of the two pyrimidine nucleobases, only thymine (or uracil) is readily deprotonated to yield a negatively charged nitrogen ligand with a significantly greater affinity for metal ions. Indeed, Hg II has been shown to selectively bind to a T-T mispair within a double-stranded oligonucleotide by coordinating to the N3 of both thymine residues [16][17][18][19]. The fact that no selectivity for uracil is observed with the 2 -O-methyl-RNA homoduplexes may be reasoned if the bulky dimethylpyrazole moieties cause a large enough steric hindrance to prevent the metallo-base pair from assuming planar geometry. Presumably, a nonplanar base pair should be more easily accommodated within the more flexible 2 -O-methyl-RNA/DNA heteroduplex than the relatively rigid 2 -O-methyl-RNA homoduplex.

Conclusions
A 2 -O-methyl-RNA oligonucleotide incorporating an artificial nucleobase that serves as a tridentate chelate for soft metal ions has been synthesized. When hybridized with a complementary DNA oligonucleotide, this modified oligonucleotide selectively recognizes a thymine residue opposite to the artificial nucleobase. The discrimination is somewhat more pronounced in the presence of 1 eq. of Pd 2+ , but the difference is too small to be taken as compelling evidence for the formation of a metal-ion-mediated base pair between thymidine and 1. Within fully matched 2 -O-methyl-RNA homoduplexes, the modification is destabilizing regardless of the presence of Pd 2+ , probably because the proposed metallo-base pair cannot assume a planar geometry needed to fit within the relatively tight base stack of these oligonucleotides. Within mismatched duplexes, as well as 2 -O-methyl-RNA/DNA heteroduplexes, the modification is stabilizing but the effect is rather sequence and Pd 2+ independent, suggesting that the origin of this stabilization is the large π-π stacking surface of the artificial nucleobase.
The metal-ion-carrying nucleobase presented in this study represents a reference structure with no potential of specific interactions with the natural nucleobases and the preference for thymine is probably based on its small size and ability to serve as an anionic nitrogen ligand. The fact that the strong base pairing observed at the monomeric level is only modestly reflected in the thermal stability of the respective double-stranded oligonucleotide is most likely due to difficulties in accommodating this (presumably) nonplanar base pair within an oligonucleotide environment. It should be noted that the CD and T m studies do not provide first-hand information on the interactions (if any) between thymine (or uracil) and the Pd 2+ complex of the artificial nucleoside 1. In other words, the possibility that within a double-stranded oligonucleotide, a complex of a different geometry prevails cannot be completely ruled out. More elaborate structures featuring additional hydrogen bonding interactions as well as carefully placed steric constraints will, in all likelihood, exhibit greatly enhanced affinity and selectivity. For example, replacing the dimethylpyrazole moieties with monosubstituted hydrazines would result in a structure not only sterically less hindered than 1 but also capable of donating two hydrogen bonds to the oxo substituents of thymine or uracil. On the other hand, using a pyrimidine, rather than a purine, nucleoside as the parent compound would alleviate some of the steric crowding caused by the relatively bulky Pd 2+ ion. Finally, other soft metal ions could be screened to find the optimal combination of affinity and selectivity.

Experimental
5.1. General. 2 ,3 ,5 -Tri-O-acetyl-6-chloro-2-iodopurine riboside was a commercial product that was used as received. For the triethylammonium acetate buffer used in HPLC chromatography, triethylamine was distilled before using. The NMR spectra were recorded with a Bruker Avance 500 NMR spectrometer and the chemical shifts are given in ppm. The mass spectra were recorded with a Bruker micrOTOF-Q ESI-MS spectrometer and the CD spectra with an Applied Photophysics Chirascan spectropolarimeter. The

9-(β-d-Ribofuranosyl)-2,6-dihydrazinopurine (3).
To neat hydrazine hydrate (6.5 mL, 134 mmol) was added 2 , 3 , 5 -tri-O-acetyl-6-chloro-2-iodopurine riboside (1.03 g, 1.91 mmol) and the resulting mixture was stirred for 14 d at room temperature. Over the course of the first day, the appearance of the reaction mixture changed from yellowish and cloudy to clear and colorless to light red, followed by formation of a white precipitate. The reaction mixture was diluted with 2-propanol (15 mL) and stirred for 15 min, after which the precipitate was collected by filtration and washed with cold 2-propanol (2 × 100 mL). Finally, the precipitate was dried under vacuum to yield 0.534 g (89%) of 2 as a white powder. 1 (3,5-dimethylpyrazol-1-yl) purine (1). To dry compound 3 (0.337 g, 1.08 mmol) was added 6.4 eq. of dry 2,4-pentanedione (0.70 mL, 6.912 mmol) and 0.1 eq. of trifluoroacetic acid (8.27 μL, 0.108 mmol). The reaction mixture was stirred at room temperature for 16 h, during which time the product crystallized. The volatiles were removed under reduced pressure to yield crude 1 that was used in the next step without further purification. For the NMR titrations, a small sample was purified by silica gel chromatography eluting with a mixture of MeOH, CH 2 Cl 2 and Et 3 N (9 : 90 : 1, v/v). 1

Oligonucleotide
Synthesis. The oligonucleotides were assembled on a CPG-supported succinyl linker at a loading of 27 mmol g −1 . Standard phosphoramidite strategies for RNA (600 s coupling time) or DNA (20 s coupling time) were used throughout the sequences, except for the modified phosphoramidite building block 2, which was coupled manually using an increased coupling time (60 min). Based on trityl response, coupling yield for building block 2 was 36% and the other couplings proceeded with normal efficiency. The products were released from support and deprotected by conventional treatment with 33% aq. NH 3 (5 hours at 55 • C). The crude oligonucleotides were purified by RP-HPLC on a Hypersil ODS column C18 (250 × 4.6 mm, 5 μm) eluting with a mixture of 0.10 mol L −1 aq. triethylammonium acetate and MeCN, the flow rate being 1.0 mL min −1 . The amount of MeCN was increased linearly from 10 to 40% during 25 min for the 2 -O-methyl-RNA oligonucleotides and from 10 to 30% during 25 min for the DNA oligonucleotides. The purified products were characterized by ESI-MS analysis.