Simon F Eskildsen

Pierrick Coupé

Kelvin K Leung

Vladimir Fonov

Nicolas Guizard

Shafik N Wassef

Lasse Riis Østergaard

D Louis Collins

BEaST: Brain extraction using multiresolution nonlocal segmentation

Keywords: Brain extraction, skull stripping, patch-based segmentation, MRI, BET

Brain extraction is an important step in the analysis of brain images. Variability in brain morphology and intensity characteristics due to different imaging sequences makes the development of a general purpose brain extraction algorithm challenging. Purpose: To address this issue, we propose a new robust method (BEaST) for brain extraction. Methods: The method is based on nonlocal segmentation embedded in a multiresolution framework. A library of 50 priors are semi-automatically constructed from the NIHPD, ICBM, and ADNI databases. Results: A mean Dice coefficient of 0.9834±0.0053 is obtained when performing leave-one-out cross validation. Validation using the online available Segmentation Validation Engine resulted in a top ranking position with a mean Dice coefficient of 0.9781±0.0047. Conclusions: The segmentation accuracy of the method is comparable to that of a recent label fusion approach, while being 40 times faster and requiring a much smaller library of priors.

Introduction

Brain extraction (or skull stripping) is an important step in many neuroimaging analyses, such as registration, tissue classification, and segmentation. While methods such as the estimation of intensity normalization fields and registration do not require perfect brain masks, other methods such as measuring cortical thickness rely on accurate brain extraction to work properly. In cases of incorrect brain extraction, subjects may be excluded from further processing, a potentially expensive consequence for most studies. The solution of manually correcting the brain masks is a labor intensive and time consuming task.

An accurate brain extraction method should separate all tissue external to the brain, such as skull, dura, and eyes, from the brain tissue without removing ⋆ Data used in the preparation of this article were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database (www.loni.ucla.edu/ADNI). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http:/loni.ucla.edu/ADNI/Collaboration/ADNI Authorship list.pdf any part of the brain. For instance, failure to remove the dura may lead to an overestimation of cortical thickness [START_REF] Van Der Kouwe | Brain morphometry with multiecho MPRAGE[END_REF], while removing part of the brain would lead to an underestimation. Several brain extraction methods have been proposed during the last decade [START_REF] Lemieux | Fast, accurate, and reproducible automatic segmentation of the brain in T1-weighted volume MRI data[END_REF][START_REF] Shattuck | Magnetic resonance image tissue classification using a partial volume model[END_REF][START_REF] Smith | Fast Robust Automated Brain Extraction[END_REF][START_REF] Ségonne | A hybrid approach to the skull stripping problem in MRI[END_REF][START_REF] Zhuang | Skull-stripping magnetic resonance brain images using a model-based level set[END_REF][START_REF] Sadananthan | Skull stripping using graph cuts[END_REF], and studies evaluating these methods have found varying accuracy [START_REF] Fennema-Notestine | Quantitative evaluation of automated skull-stripping methods applied to contemporary and legacy images: Effects of diagnosis, bias correction, and slice location[END_REF][START_REF] Shattuck | Online resource for validation of brain segmentation methods[END_REF]. While some methods are better at removing non-brain tissue, at the cost of removing brain tissue, others are better at including all brain tissue, at the cost of including non-brain tissue [START_REF] Fennema-Notestine | Quantitative evaluation of automated skull-stripping methods applied to contemporary and legacy images: Effects of diagnosis, bias correction, and slice location[END_REF][START_REF] Shattuck | Online resource for validation of brain segmentation methods[END_REF] -classical example of sensitivity vs. specificity. A method that works reliably and robustly on a variety of different brain morphologies and acquisition sequences without requiring adjustment of parameters would greatly reduce the need for manual intervention and exclusion of subjects in neuroimaging studies.

Building on recent work on label fusion [START_REF] Collins | Towards accurate, automatic segmentation of the hippocampus and amygdala from MRI by augmenting ANIMAL with a template library and label fusion[END_REF], multi-atlas propagation and segmentation (MAPS) [START_REF] Leung | Automated cross-sectional and longitudinal hippocampal volume measurement in mild cognitive impairment and Alzheimer's disease[END_REF] was adapted to brain extraction to address the problem of variability in anatomy and acquisition, producing more robust results [START_REF] Leung | Brain MAPS: An automated, accurate and robust brain extraction technique using a template library[END_REF]. In label fusion, multiple atlases are selected from a library of labeled images and, by means of nonrigid registration, merged in the target image to obtain a segmentation. The method is highly dependent on the accuracy of the nonrigid registrations. Registration errors may result in later segmentation errors, as all selected labels typically are weighted equally. Due to the time consuming multiple nonrigid registrations step in MAPS, the processing time per subject on an Intel R Xeon R CPU (3GHz) is 19 h.

A recent framework inspired by nonlocal means filtering [START_REF] Coupé | An Optimized Blockwise Nonlocal Means Denoising Filter for 3-D Magnetic Resonance Images[END_REF] has been introduced to achieve the label fusion task. This method has demonstrated promising segmentation results without the need for nonrigid registrations [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF]. Instead of performing the fusion of nonlinearly deformed template structures, this method achieves the labeling of each voxel individually by comparing its surrounding neighbourhood with patches in training subjects in which the labels of the central voxels are known. The patch-based segmentation method cannot be directly applied to brain extraction, because i) false positives are likely to occur as extracerebral tissue may resemble brain within the patch structure, and ii) the computational complexity is high and becomes a problem for large structures. To address these issues, we propose to apply a multiresolution approach to the patch based segmentation for performing brain extraction.

Methods

The proposed method, Brain Extraction based on nonlocal Segmentation Technique (BEaST), is inspired by the patch-based segmentation published in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF]. As done in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF], we use sum of squared differences (SSD) as the metric for distance between patches. Thus, we perform intensity normalization and spatial normalization before constructing the library of priors. The gold standard of library priors is constructed using a semi-automatic method that involves manual correction of the brain masks, because manual brain segmentation from scratch is a time consuming process.

The following describes the normalization, construction of the library containing the priors, and fundamental patch-based segmentation method as well as our contribution of embedding the method in a multiresolution approach to improve segmentation accuracy and computation time. Our main contribution is the development of a robust procedure to identify accurate brain masks with an extensive validation on multiple datasets acquired on different scanners.

Normalization

Image intensity normalization is performed by first applying the bias field correction algorithm N3 [START_REF] Sled | A nonparametric method for automatic correction of intensity nonuniformity in mri data[END_REF] followed by a linear intensity normalization, which scales the intensities to the range [0;100] using 0.1%-99.9% of the voxels in the intensity histogram. Spatial normalization is done by 9-parameter linear registration to the publicly available ICBM152 average [START_REF] Fonov | the Brain Development Cooperative Group: Unbiased average ageappropriate atlases for pediatric studies[END_REF] (stereotaxic space=193×229×193 with isotropic 1 mm voxels).

Definition of brain mask

Our goal for the brain extraction is to include all grey matter (GM) and white matter (WM) as well as internal CSF and some external CSF. Thus ventricles and deep sulci should be included in the resulting segmentation while all dura, exterior blood vessels, and nerves should be excluded. This definition is similar to results produced by the popular Brain Extraction Toolkit (BET) [START_REF] Smith | Fast Robust Automated Brain Extraction[END_REF], except we aim at consistently excluding the superior sagittal and transverse sinuses and the optic chiasms, which in most cases are included in BET segmentations. Furthermore, BET sometimes includes dura proximate to the brain, caratid arteries, ocular fat / muscle, and parts of the eyes, while cutting off gyri in cases of atrophied brains. Our aim is to eliminate such errors in a consistent manner.

Construction of library priors

The library of segmentation priors is built from data in the NIH Pediatric Database (NIHPD) [START_REF] Evans | The NIH MRI study of normal brain development[END_REF] (age: 5-18y), the International Consortium for Brain Mapping (ICBM) database [START_REF] Mazziotta | A Probabilistic Atlas of the Human Brain: Theory and Rationale for Its Development : The International Consortium for Brain Mapping (ICBM)[END_REF] (age: 18-43y), and the Alzheimer's Disease Neuroimaging Initiative (ADNI) database [START_REF] Mueller | The Alzheimer's Disease Neuroimaging Initiative[END_REF] (age: 55-91y). The NIHPD and ICBM databases consist of healthy subjects, while the ADNI database, in addition to cognitive normal (CN) subjects, contains scans of subjects with Alzheimer's disease (AD) and mild cognitive impairment (MCI). This way the entire human life span is almost covered and subjects with atrophic morphology are included, which provides a representative library of priors for performing brain extraction.

We chose 10 random T1-weighted (T1w) magnetic resonance (MR) scans from each of the NIHPD and ICBM databases. From the ADNI database we chose 10 random T1w MR scans at the baseline timepoint from each class (CN, MCI, AD). In total, our library consists of 50 priors.

The NIHPD and ICBM databases contain T2w and PDw images in addition to T1w images. T1w images have high signal for the brain tissue, while T2w and PDw images have high signal for cerebro-spinal fluid (CSF) (see Fig. 1). We utilize this in defining the library priors. By adding intensities from the three different sequences we obtain an image with a very high signal for the intracranial cavity (ICC), which can be easily extracted using BET. From the ICC segmentation we use Fast Accurate Cortex Extraction (FACE) [START_REF] Eskildsen | Active Surface Approach for Extraction of the Human Cerebral Cortex from MRI[END_REF] to delineate the boundary between GM and CSF in the cerebrum. Cerebellum and brain stem are added by non-linearly fitting masks in stereotaxic space. Finally, extensive and careful manual corrections were performed to get an optimal brain segmentation matching our definition. On average, such corrections took between 1-2 hours per brain.

Priors from the ADNI database are constructed using the semi-automatic segmentations used in MAPS [START_REF] Leung | Brain MAPS: An automated, accurate and robust brain extraction technique using a template library[END_REF], which are publicly available at the ADNI website. These segmentations are accurate definitions of the GM and WM of the brain, but all interior CSF is excluded (see Fig 2). Therefore, we deform a spherical mesh initialized around the brain to fit smoothly along the border of the segmentation. This way we obtain a similar definition of a brain segmentation as for the NIHPD and ICBM data. Finally, these segmentations were manually corrected in the same way as the NIHPD and ICBM data.

All library priors were flipped along the midsagittal plane to increase the size of the library utilizing the symmetric properties of the human brain.

Patch-based segmentation

We use the patch-based segmentation method described in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF]. In brief, a label is applied to a given voxel in the target image based on the similarity of its surrounding patch P (x i ) to all the patches P (x s,j ) in the library. For each voxel x i of the target image, the surrounding neighbourhood V i is searched for similar patches in the N library images. A nonlocal means estimator v(x i ) is used to estimate the label at x i :

v(x i ) = N s=1 j∈Vi w(x i , x s,j )l(x s,j ) N s=1 j∈Vi w(x i , x s,j ) , (1)
where l(x s,j ) is the label of voxel x s,j at location j in library image s. We used l(x s,j ) ∈ {0, 1}, where 0 is background and 1 is object (brain). The weight w(x i , x s,j ) assigned to label l(x s,j ) depends on the similarity of P (x i ) to P (x s,j ) and is computed as:

w(x i , x s,j ) = e -P (x i ,P (x s,j )) 2 2 h 2 ,
(2) where ||•|| 2 is the L2-norm, normalized by the number of elements and computed between each intensity of the elements of the patches P (x i ) and P (x s,j ). The parameter h of the weighting function is locally adapted as in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF].

Computation time rises quickly if these calculations are to be made for all patches in all library images. To decrease computation time, the N closest images from the library are selected based on their similarity to the target image within a defined region of interest (ROI) (initialization mask, see eq. 3). The similarity is calculated as the SSD between the target and each library image. Furthermore, to reduce the number of patches to consider, preselection of patches is done as proposed in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF]. Finally, an initialization mask M is constructed as the union of all segmentation priors S i minus the intersection of all S i :

M = (S 1 ∪ S 2 ∪ ... ∪ S N )\(S 1 ∩ S 2 ∩ ... ∩ S N ).
(

) 3
The patch-based segmentation is performed within this ROI only under the assumption that the library is representative of all brain sizes after spatial normalization. This approach reduces the ROI by 50% compared with the union of all S i and by 85% compared with the entire stereotaxic space (Fig. 3).

Multiresolution framework

In order to obtain optimal performance for brain extraction, the patch size needs to be large compared to the patch sizes used for smaller structures, such as the hippocampus. A large patch size is needed to avoid inclusion of extra-cerebral tissue, such as dura. This is computationally impractical in the native resolution. Therefore, we suggest to embed the patch based segmentation in a multiresolution framework, which provides the opportunity to have large patch sizes while still being computationally practical.

In brief, the multiresolution framework enables propagation of segmentation across scale by using the resulting segmentation at the previous scale to initialize the segmentation at the current one.

The library images, labels, initialization mask, and target image at the stereotaxic resolution V j are all resampled to a lower resolution V j-k , and the patch-Fig. 2. Adaptation of library priors using deformable surface. A) Semi-automatic GM/WM mask as used by MAPS [START_REF] Leung | Brain MAPS: An automated, accurate and robust brain extraction technique using a template library[END_REF]. B) Adapted mask generated by deforming a surface mesh to the boundary of the GM/WM mask and manually corrected.

based segmentation is performed. The nonlocal means estimator v j-k V (x i ) at the V j-k resolution is propagated to a higher resolution V j-k+1 by upsampling using trilinear interpolation. The estimator function v j-k V (x i ) can be considered as the confidence level of which label to assign the voxel. Values close to 0 are likely background, while values close to 1 are likely object. We define a confidence level α to assign labels to the voxels at each scale. Voxels with v j-k V (x i ) < α are labelled background, and voxels with v j-k V (x i ) > (1α) are labelled object. Segmentation of these voxels is considered final, and they are excluded from further processing. Voxels with v j-k V (x i ) in the range [α; 1α] are propagated and processed at a higher resolution (V j-k+1 ). This procedure is repeated until the resolution of the stereotaxic space V j is reached. This way the initialization mask of each resolution step is limited to the voxels with uncertain segmentation at the previous step (Fig. 3). This greatly reduces the computational cost. At the stereotaxic resolution, segmentation is done by thresholding the estimator v j V (x i ) at 0.5.

During experiments, we used three resolutions (k = 2) with isotropic voxel sizes respectively of 4 mm, 2 mm, and 1 mm (stereotaxic space resolution) (see Fig. 3). We empirically chose confidence level α and variable patch size and search area depending on the resolution (see table 1).

Voxel Size (mm) Patch Size Search Area α 4×4×4 3×3×3 3×3×3 0.2 2×2×2 3×3×3 9×9×9 0.2 1×1×1
5×5×5 13×13×13 -Table 1. Patch size, search area, and confidence level α chosen for the three resolutions.

Results

We measured the segmentation accuracy in a leave-one-out cross validation (LOOCV) fashion. Each of the 50 library images was processed with the re-maining 49 images as priors, and the resulting segmentation was compared to manually corrected labels in the library. Fig. 4 shows the Dice coefficients for increasing number of priors selected from the library. As shown in [START_REF] Coupé | Patch-based segmentation using expert priors: Application to hippocampus and ventricle segmentation[END_REF], increasing the number of selected priors improves the segmentation accuracy. In our experiment, accuracy is high even when using only very few selected priors. Increasing the number of selected priors seems to make the segmentations more consistent. We chose N = 20 for further experiments. Fig. 6(a) shows the segmentation accuracy within the different groups used in the experiment. The accuracy on ICBM data is significantly higher than the accuracy on the other groups tested. This may be due to the fact that ICBM data are more homogeneous than the other groups, which lead to higher redundancy and better matches of patches during the segmentation process.

A comparison to BET was performed by running BET with the optimal parameters reported in [START_REF] Leung | Brain MAPS: An automated, accurate and robust brain extraction technique using a template library[END_REF]. Fig. 5 shows typical examples of brains masks obtained by BET and BEaST on the five different groups tested here (NIHPD, ICBM, ADNI-CN, ADNI-MCI, ADNI-AD). On NIHPD and ICBM data, BET behaves quite well with only minor segmentation errors, such as inclusion of the transverse sinus and part of the eye sockets. On ADNI data, more serious errors are found using BET. These include inclusion of dura and cartilage while gyri are often cut off in atrophic brains. BEaST generally provides a more consistent and robust segmentation without serious errors.

We measured the segmentation output at each resolution by thresholding the nonlocal means estimator at 0.5. Fig. 6(b) shows the resulting Dice coefficient compared to BET. As shown, the accuracy increases along with scale, and at 2 mm voxel sizes (requiring about 1.2 minutes) BEaST has higher accuracy than BET. The difference in Dice coefficients may seem small. However, when measuring Dice coefficients in the context of whole brain segmentations, small We validated BEaST using an independent test set. Validation was performed using the online Segmentation Validation Engine (SVE) of brain segmentation methods [START_REF] Shattuck | Online resource for validation of brain segmentation methods[END_REF]. The web service allows the comparison of results with 40 handcorrected brain masks. The website contains an archive of all uploaded results, which enables segmentation methods to be objectively benchmarked and compared. Images from the independent test set were normalized the same way as the library images. Validation using the test set resulted in a mean Dice coefficient of 0.9781±0.0047 (see http://sve.loni.ucla.edu/archive/study/?id=244). At the time of writing, this result was the best of all the methods published on the website. MAPS had a second place with a Dice coefficient of 0.9767±0.0021. When compared with BEaST, these results are statistically significant (Student's group t-test, p=0.05). Futhermore, MAPS requires 19 hours of computation time, compared to 30 minutes for BEaST on similar hardware.

Computation time

In our experiments with 20 images selected from the library, the total processing time on an Intel R i7 R processor at 3 GHz was less than 30 min per subject. With 10 images, the processing time was less than 20 min per subject. By contrast, without the multiresolution step and using the initialization mask, the processing time was around 320 min. Removing the initialization mask increased the processing time to 42 h.

The average processing time of BET was about 2 minutes. Obtaining the segmentation of BEaST at 2 mm voxel sizes takes about 1.25 minutes and the corresponding Dice coefficients are higher than BET. This suggests that a fast low resolution result is in many cases sufficient for the subsequent analysis.

Discussion

The leave-one-out cross validation showed that the segmentation accuracy is consistently high (average Dice coefficient: 0.9834±0.0053) and that selecting more than 10 priors does not noticeably increase the accuracy. Our results are similar to recent ones from a label fusion approach [START_REF] Leung | Brain MAPS: An automated, accurate and robust brain extraction technique using a template library[END_REF], where the library is almost 14 times larger and the processing time, about 40 times longer. The short processing time in BEaST (<30 min) results from only needing linear registrations and the advantage of using the ROI in the multiresolution strategy. The current implementation runs as a single thread. However, the nonlocal means calculations can easily be parallelized and implemented to exploit GPU processing [START_REF] De Fontes | Real time ultrasound image denoising[END_REF], which will decrease processing time significantly.

Compared to the widely used brain extraction method, BET, BEaST more than halfs the segmentation error increasing the average Dice coefficient from 0.9646 to 0.9834. In terms of speed, BET is faster than BEaST, if the segmentations are performed at the highest resolution. However, stopping the processing at 2 mm voxel sizes results in computation times similar to BET, while still obtaining higher segmentation accuracy.

The results showed a higher accuracy on ICBM data. This may be caused by the fact that the anatomical variability within this group is smaller than the other groups studied. This suggests that the accuracy may be improved by extending the number of priors for the groups with higher anatomical variability. Though the results show that only a relatively small library is needed, the library still needs to be representative for the patch-based segmentation to work optimal.

Using the online segmentation validation engine [START_REF] Shattuck | Online resource for validation of brain segmentation methods[END_REF] we obtained a truly objective measure of the performance of BEaST. A mean Dice coefficient of 0.9781 is significantly better than the best score by MAPS (0.9767). Compared to MAPS, a relatively small number of library priors are needed in the patch-based segmentation. This makes it feasible to distribute the method as downloadable software. We intend to make BEaST available online if permission to redistribute the data in the library can be obtained.

As in label fusion, the nonlocal segmentation approach enables the segmentation of different targets simultaneously. For example, the intracranial cavity may be obtained by generating priors using appropriate methods, such as the multi-modal approach used as intermediate step to obtain the brain segmentation priors. Also, separation of cerebellum and brain stem from the cerebrum may be achieved with high accuracy if the structural priors are available.

In conclusion, we have proposed a new brain extraction method, BEaST, based on nonlocal segmentation embedded in a multiresolution framework. The accuracy of the method is higher than BET and similar to that of a recent label fusion method, while being much faster and requiring a smaller library of priors. brain segmentations. KKL acknoweldges support from the MRC, ARUK and the NIHR.

Fig. 1 .

 1 Fig. 1. Construction of library priors using multiple modalities. A) Intensities from T1w, T2w, and PDw images are added. B) BET is used to produce an ICC mask. C) FACE is used to delineate the cortical boundary and produce a cerebrum mask. D) Cerebellum and brain stem are added by stereotaxic masks, and the mask is manually corrected.

Fig. 3 .

 3 Fig. 3. The multiresolution segmentation process (row 1-3) compared to a single resolution approach (row 4). Column 1: Initialization mask. Column 2: Nonlocal means estimator map. Column 3: Segmentation by thresholding the NLM estimator and adding the intersection mask. Processing times are accumulated time from initialization. Notice the inclusion of dura in the single resolution approach.

Fig. 4 .

 4 Fig. 4. Dice coefficient of segmentations using an increasing number of priors from the library. Experiment performed by leave-one-out.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Typical results using BET and BEaST on the five test groups. Column 1: Manual segmentation. Column 2: BET segmentation. Column 3: BEaST segmentation. Blue voxels are overlapping voxels in the segmentation compared to the gold standard.Green voxels are false positives and red voxels are false negatives. Generally, BEaST consistently removes dura, sinuses and optic chiasms, while this is not the case for BET.

Acknowledgements

The authors would like to thank Professor Nick Fox, Dementia Research Centre, Institute of Neurology, London, for contributing with the ADNI semi-automatic


Vilve Nummert
email: 1vilve.nummert@ut.ee

Vahur Mäemets
email: 2vahur.maemets@ut.ee

Mare Piirsalu
email: 3mare.piirsalu@ut.ee

Ilmar A Koppel
email: 4ilmar.koppel@ut.ee

Influence of ortho substituents on 17 O NMR chemical shifts in phenyl esters of substituted benzoic acids

Keywords: NMR spectra of phenyl benzoates, 17O NMR chemical shifts, substituent effects, ortho effect, correlation equations NMR spectra of phenyl benzoates, 17 O NMR chemical shifts, substituent effects, ortho effect, correlation equations http://mc.manuscriptcentral.com/poc

17 O NMR spectra for 29 phenyl esters of ortho-, para-and meta-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , at natural abundance in acetonitrile were recorded. The δ( 17 O) values of carbonyl and the single-bonded oxygens for para derivatives gave good correlation with the σ + constants. The δ( 17 O) values for meta derivatives correlated well with the σ m constants. The influence of ortho substituents on the δ( 17 O) values of carbonyl oxygen and the singlebonded oxygens was analyzed using the Charton equation containing the inductive, σ I , resonance, σ + R , and steric, E s B , substituent constants. For ortho derivatives excellent correlations with the Charton equation were obtained when the data treatment was performed separately for derivatives containing electron-donating +R and electronattracting -R substituents. The electron-donating substituents in ortho-, meta-and parasubstituted esters resulted in shielding of the 17 O signal and the electron-withdrawing groups caused deshielding. In phenyl ortho-substituted benzoates the substituent-induced positive inductive (ρ I > 0), resonance (ρ R > 0) and steric (δ ortho E s B > 0) effects were found. The steric interaction of ortho substituents with ester group was found to produce a deshielding effect on the carbonyl and single-bonded oxygens. For ortho derivatives with -R substituents the resonance term was insignificant and the steric term was ca. twice weaker as compared to that for derivatives with +R substituents. The δ( 17 O) values for ortho-substituted nitrobenzenes, acetophenones and benzoyl chlorides showed a good correlation with the Charton equation as well. In ortho-substituted nitrobenzenes the inductive, resonance and steric effect were found to be ca. 1.7 times stronger as compared to that for phenyl ortho-substituted benzoates.

INTRODUCTION

In our previous papers the influence of substituent effects, especially ortho effect, on the rates of the alkaline hydrolysis in various media (as shown in References [1][2][3][4][5][6][7] ), the carbonyl carbon 13 C NMR chemical shifts, δ CO , [8] and the carbonyl carbon infrared stretching frequencies, ν CO , [9] in substituted phenyl benzoates, (X-C 6 H 4 CO 2 C 6 H 5 , C 6 H 5 CO 2 C 6 H 4 -X) were studied. In the present paper we extend our study of the ortho substituent effect to the To the best of our knowledge, no study of the ortho, meta, and para substituent effects on the δ( 17 O) values for phenyl esters of substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , could be found in the literature. There are also no δ( 17 O) data for the phenyl esters of substituted benzoic acids, except unsubstituted phenyl benzoate, X = H, [10,11] available in the literature.

The values of δ( 17 O) in the ortho-substituted esters of substituted benzoic acids are available only for methyl benzoates, X-C 6 H 4 CO 2 CH 3 , with X = OCH 3 , Cl, NO 2 , CH 3 and 2,6-disubstituted methyl benzoates, X 1 X 2 -C 6 H 4 CO 2 CH 3 , with X 1 = X 2 = CH 3 , C(CH 3 ) 3.

[ 12-14] Although there are numerous investigations on the δ( 17 O) values for esters in literature, [10-33] the influence of the substituent effects on the δ( 17 O of the carbonyl and methoxy oxygens in esters of substituted benzoic acid, using correlation equations, has been studied only for meta-and para-substituted methyl benzoates and para-substituted methyl 2,6-dimethyl-4-X-benzoates. [14][15][16] The δ( 17 O) values of carbonyl and methoxy oxygens in meta-and para-substituted methyl benzoates gave good correlations with σ m and σ + constants, respectively. In the hindered benzoates, 2,6-dimethyl-4-X-benzoates, the through-conjugation was found to be essentially reduced due to the increased torsion angle between the carbonyl group and the ring plane. [13,14] It was found that in methyl benzoates reaction series an introduction of electron-attracting substituents causes deshielding and electron-donating substituents shielding of the carbonyl and methoxy O atoms. [14][15][16] It has been shown [26][27][28][29] that in substituted carbonyl derivatives, Y-C 6 H 4 CO-X, RCO-X, the shielding of the carbonyl oxygen is increased and the sensitivity of the Y substituent is diminished with an increase in the electron-donating power of the X group.

In ortho-substituted compounds the δ( 17 O) data have been quite extensively studied for nitrobenzenes, [34,35] acetophenones, [36][37][38] benzaldehydes, [36] and benzoyl chlorides. [39][40] But to the best of our knowledge, up to now the correlation equations were not applied to describe the influence of ortho substituents on the 17 O NMR chemical shifts. In orthosubstituted derivatives the steric influence of ortho substituents on the δ( 17 O) values has been explained by steric inhibition of resonance and van der Waals interactions. [12-14,17,39,41- 44] It was suggested that in systems in which the functional group is free to rotate, due to sterical consequences the functional group is rotated out of the plane of the aromatic ring increasing the double bond character of the functional group. In the rigid systems the steric influence of ortho substituents was explained by the van der Waals interactions between the ester group and the adjacent bulky substituent. [41,42] In the previous papers we found that in substituted phenyl benzoates, (X-C 6 H 4 CO 2 C 6 H 5 , C 6 H 5 CO 2 C 6 H 4 -X) the influence of ortho substituents on the log k values in the alkaline hydrolysis, [1][2][3][4][5][6][7] the carbonyl carbon 13 C NMR chemical shifts, δ CO , [8] and the carbonyl carbon infrared stretching frequencies, ν CO , [9] was precisely described with the Charton equation [45] using the inductive, σ I , [8,46] resonance, σ°R, [8,47] and steric, E S F o r P e e r R e v i e w 3 water, [6] the carbonyl carbon infrared stretching frequencies, ν CO , [1,9] for cis conformers as well as in the 13 C NMR chemical shifts, δ CO : [8] log k ortho (H 2 O, 25 °C) = -0.333 + 2.13σ I + 0.31σ°R + 2. In reaction series considered (Eqns (1)-( 2)) the resonance term was negligible. In the 13 C NMR chemical shifts, δ CO , [8] the value of (ρ R ) ortho has the opposite sign ((ρ R ) ortho = +1.6) as compared to that for para derivatives ((ρ R ) para = -1.00).

Recently we found [1,8] good correlations between the log k values of the alkaline hydrolysis of ortho-substituted phenyl benzoates and the infrared stretching frequencies of carbonyl group, ν CO , as well as the 13 C NMR chemical shifts, δ CO , in case the additional resonance and steric scales were included.

The aim of the present paper was to check the applicability of the Charton equation to describe the influence of ortho substituents on the δ( 17 O) values in phenyl esters of orthosubstituted benzoic acids, (X-C 6 H 4 CO 2 C 6 H 5 ), and to compare the substituent effects in the δ( 17 O) values with those in the alkaline hydrolysis, [6] the infrared stretching frequencies, ν CO , [9] and the 13 C NMR chemical shifts, δ CO . [8] For the comparison, the δ( 17 O) values for substituted nitrobenzenes, [34,35] acetophenones, [36,38] and benzoyl chlorides, [39][40] published in literature were correlated with the Charton equation as well.

EXPERIMENTAL NMR measurements

17 O NMR spectra for 29 phenyl esters of ortho-, meta-and para-substituted benzoic acids,

X-C 6 H 4 CO 2 C 6 H 5 (X = H, 4-NO 2 , 4-F, 4-Cl, 4-Br, 4-CH 3 , 4-OCH 3 , 4-NH 2 , 4-C(CH 3 ) 3 , 3- NO 2 , 3-CN, 3-Cl, 3-F, 3-CH 3 , 3-OCH 3 , 3-N(CH 3 ) 2 , 2-NO 2 , 2-CN, 2-F, 2-Cl, 2-Br, 2-I, 2- CF 3 , 2-CH 3 , 2-OCH 3 , 2-N(CH 3 ) 2 , 2-NH 2 , 2-SO 2 CH 3 , 2-SCF 3 )
, were recorded at natural abundance in acetonitrile (see 4 was acquired at the spectral width 23148 Hz and its size 2048 data points. The 10 µs (at the ~11µs 90 degree pulse) exitation pulse with pre-scan delay 64 µs and with the relaxation delay 50 ms was applied. Acquisition time was 44 ms. The data were processed with Bruker TopSpin 2.0 software package. During the data processing an exponential multiplication of the FID with LB = 40 was applied. For the correction of the baseline rolling caused by acoustic ringing effect a backward linear prediction for the first 10 data points of the FID was applied also during of the data processing. The line width of the 17 O spectral lines of the phenyl esters measured was within the range 250-400 Hz and up to 50-60 Hz for the water standard. The error of the chemical shifts measured was estimated on the basis of repetitive measurements and was ≤ ±0.4 ppm.

The standard 1 H and proton-decoupled 13 C NMR spectra were recorded in CDCl 3 . solution with 1% TMS added for the spectral referencing at a room temperature and at 200.13 and 50.33 MHz correspondingly. 2D HSQC or HETCOR spectra were recorded also for assignment of the 1 H and 13 C peaks.

Synthesis of compounds

The preparation procedure and characteristics for the phenyl esters of the ortho-, meta-and para-substituted benzoic acids, (X-C 6 H 4 CO 2 C 6 H 5 ), has been described in Reference. [6] The phenyl esters of 2-N(CH 3 ) 2 -, 2-SO 2 CH 3 -and 2-SCF 3 -substituted benzoic acids were prepared by the addition of thionyl chloride to the mixture of corresponding orthosubstituted benzoic acid and phenol in pyridine at 0 ºC with stirring. [50] Phenyl 2-N(CH 3 ) 2benzoate was recrystallized from aqueous 80% ethanol: yield 62%, m.p. 63-64 ºC, Reference, [51] m.p. 67-69 ºC. Phenyl 2-SO 2 CH 3 -benzoate was recrystallized from aqueous 67% ethanol: yield 56%, m.p. 116-117 ºC, Reference, [51] m.p. 108-110 ºC. Phenyl 2-SCF 3benzoate was extracted from the reaction mixture with diethyl ether. The ether layer was dried with unhydrous magnesium sulfate. Diethyl ether was removed under the reduced pressure. Phenyl 2-(trifluoromethylthio)benzoate: b.p. 145-147ºC/2.5 mbar, yield 40%. The phenyl esters of 3-CN-, 3-OCH 3 -and 3-F-substituted benzoic acids were prepared from corresponding substituted benzoyl chloride and phenol in pyridine solution. [52] Phenyl 3-CN-benzoate was recrystallized from aqueous ethanol: yield 27%, m.p. 95-96 ºC, Reference, [53] m.p. 92-95. Phenyl 3-OCH 3 -benzoate: yield 66%, m.p. 62-63, Reference, [53] m.p. 64-66 ºC. Phenyl 3-F-benzoate: yield 64%, m.p. 55-56, Reference, [54] 1 H NMR: 8.50 dt, 1H (H-2), 4 J aver = 1.7, 5 J = 0.7; 7.92 ddd, 1H (H-4), 3 J = 7.8, 4 J =1.7 and 1.3; 7.67 dt, 1H (H-5), 3 J aver = 7.9, 5 J = 0.7; 8.43 ddd, 1H (H-6), 3 J = 7.9, 4 J = 1.3 and 1.7, 7.18-7.35 m, 3H (H-9,11,13); 7.46 m, 2H (H-10,12), 3 J aver = 7.9. 1 H NMR: 7.87 dddd, 1H (H-2), 3 J F-H = 9.2, 4 J =1.6 and 2.7, 5 J = 0.4; 7.16-7.52 m, 7H (H-4,5,9-13); 7.99 ddd, 1H (H-6), 3 J = 7.7, 4 J =1.6 and 1.1. The δ( 17 O) values for the carbonyl and single-bonded oxygens in the phenyl esters of paraand meta-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , given in Table 1 were correlated with Eqns (4) and (5). For meta substituents σ m , σ I and σ°R were used.

DATA PROCESSING AND RESULTS

F

δ( 17 O) para(meta) = δ( 17 O) H + (ρ + ) para(meta) σ + (σ m ) (4) δ( 17 O) para(meta) = δ( 17 O) H + (ρ I ) para(meta) σ I + (ρ + R ) para(meta) σ + R (σ°R) (5)
In the case of ortho-substituted derivatives the influence of the inductive, resonance and steric factors on the δ( 17 O) values was separated using the following Charton equations [45] :

δ( 17 O) ortho = δ( 17 O) H + (ρ I ) ortho σ I + (ρ + R ) ortho σ + R + δ ortho E s B ( 6
)
δ( 17 O) ortho = δ( 17 O) H + (ρ I ) ortho σ I + (ρ + R ) ortho σ + R + δ ortho υ (7)
The correlation of the δ( 17 O) values with Eqns ( 6) and ( 7) was performed separately for ortho-substituted derivatives containing the ortho electron-donating +R substituents (X = OCH 3 , CH 3 , N(CH 3 ) 2 , F, Cl, Br, H) and derivatives with the ortho electron-withdrawing -R substituents (X = NO 2 , CN, CF 3 , SCF 3 , SO 2 CH 3 , H). The Brown and Okamoto constants, σ + , [55,56] the polar substituent constants, σ m , [56] the Taft inductive σ I, [8,46]

resonance σ + R constants (σ + R = σ + p -σ I )
, and σ°R (σ°R = σ°p -σ I ) [8,47] were used in the data processing.

As the steric constants for ortho substituents two steric scales were employed: the E s B constants and the Charton scale of υ. [49,60,61] The steric constants, E s B , determined on the basis of the acid hydrolysis of ortho-substitued phenyl benzoates, C 6 H 5 CO 2 C 6 H 4 -X, [6,48] in the case of substituents X = H, F, Cl, Br, I, CH 3 , C(CH 3 ) 3 , CF 3 , were found to be nearly linear function of the υ values, calculated on the bases of van der Waals radii, r v .

The results of correlations of the carbonyl oxygen and the single-bonded oxygen δ( 17 O) values, for phenyl esters of para-, meta-and ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , with Eqns (4)-( 7) are listed in Table 2. For comparison, the correlation of the δ( 17 O) values published in literature for ortho-, para-and meta-substituted nitrobenzenes, [17,34,35,57,58] X-C 6 H 4 NO 2 , acetophenones, [36][37][38]59] X-C 6 H 4 COCH 3 , and benzoyl chlorides, [27,39,40] X-C 6 H 4 COCl, with Eqns ( 4)-( 6) are shown in Table 2 as well.

To compare of the substituent effects on the δ( 17 O) values of the carbonyl oxygen and the single-bonded oxygen for phenyl esters of ortho-, para-and meta-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , with those in the kinetic data of the alkaline hydrolysis, log k, the carbonyl carbon 13 C NMR chemical shifts, δ CO , and the infrared stretching frequencies of the carbonyl group, ν CO , in substituted phenyl benzoates, X-C 6 H 4 CO 2 C 6 H 5 , the following relationships were used:

δ( 17 O) ortho = δ( 17 O) H + a 1(ortho) ∆log k X + a 2(ortho) σ + R + a 3(ortho) E s B (8) δ( 17 O) ortho = δ( 17 O) H + a 1(ortho) (∆δ CO ) X + a 2(ortho) σ + R + a 3(ortho) E s B (9) F o r P e e r R e v i e w 8 δ( 17 O) ortho = δ( 17 O) H + a 1(ortho) (∆ν CO ) X + a 2(ortho) σ + R + a 3(ortho) E s B (10) δ( 17 O) para(meta) = δ( 17 O) H + a 1(para)(meta) ∆log k X + a 2(para)(meta) σ + R (σ°R) (11) δ( 17 O) para(meta) = δ( 17 O) H + a 1(para)(meta) (∆δ CO ) X + a 2(para)(meta) σ + R (σ°R) (12) δ( 17 O) para(meta) = δ( 17 O) H + a 1(para)(meta) (∆ν CO ) X + a 2(para)(meta) σ + R (σ°R) (13)
In Eqns ( 8)-( 13)

∆log k X = log k X -log k H , (∆δ CO ) X = (δ CO ) X -(δ CO ) H and (∆ν CO ) X = (ν CO ) X -(δ CO ) H .
The log k values for the alkaline hydrolysis in water and aqueous 50% DMSO, the carbonyl carbon 13 C NMR chemical shifts, δ CO , and the IR stretching frequencies of the carbonyl group, ν CO , for phenyl benzoates, X-C 6 H 4 CO 2 C 6 H 5 , used in correlations with Eqns ( 8)-( 13) are given in References. [6,8,9,51,52] In the case of phenyl esters of orthosubstituted benzoic acids, the ν CO values for cis conformers estimated by the carbonyl stretching frequencies deconvolution were used. The results of data treatment with Eqns ( 8)-( 13) are listed in Table 3. For the data processing a multiple-parameter linear leastsquares (LLSQ) procedure [62] was used.

DISCUSSION

The δ( 17 O) values in phenyl esters of para-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , (Table 1) gave excellent correlations so with the single parameter treatment using the Brown and Okamoto σ + constants [55,56] (Eqn (4)) as the dual substituent parameter treatment with the Taft σ I [8,46] and σ + R (σ + R = σ + -σ I ) constants (Eqn (5), Table 2):

δ( 17 O) para = (345.2 ± 0.3) + (9.07 ± 0.55)σ + (14) R = 0.986, s = 0.918, n/n 0 = 9/9 δ( 17 O) para = (345.9 ± 0.5) + (7.21 ± 0.99)σ I + (9.71 ± 0.54)σ + R (15) R = 0.990, s = 0.752, n/n 0 = 9/9
In para-substituted phenyl benzoates the calculated sensitivity of the δ( 17 O) values of the carbonyl oxygen towards the inductive effect and through-conjugation shown by Eqns ( 14) and ( 15) (Table 2) were approximately the same as earlier were found for methyl benzoates in References [15,16] (ρ + = 9.76, [15] 9.03, [16] 9.66 (Table 2)). The excellent correlation of the δ( 17 O) values obtained with σ + , σ I and σ + R constants (Eqns ( 14) and ( 15)) suggests that the δ( 17 O) values for phenyl esters of para-substituted benzoic acids are besides the inductive effect dependent on the through-conjugation between substituent and the carbonyl oxygen similar to that for para-substituted methyl benzoates, [14][15][16] acetophenones, [36,37,59] benzoyl chlorides, [27,39,40] nitrobenzenes. [35,57-58] The δ( 17 O) values of the single-bonded phenoxy oxygen show good correlations with Eqns (4) and ( 5 2) which is similar to that in References. [14][15][16] The chemical shifts of carbonyl oxygen for meta derivatives correlated well with the single parameter treatment using the Taft σ m constants as the dual substituent parameter treatment with the Taft σ I and σ°R constants (Table 2).

In meta-substituted phenyl benzoates the calculated sensitivity of the δ( 17 O) values of carbonyl oxygen towards the σ m constants was nearly the same as earlier found for methyl benzoates in Reference [15] (ρ m = 6.44, [15] 7.17 Table 2). In the case of meta derivatives the δ( 17 O) values for single-bonded phenoxy oxygen showed very low sensitivity towards the substituent effects in benzoyl moiety (ρ m = 1.71 Table 2). In case the δ( 17 O) values of single-bonded phenoxy oxygen for meta derivatives were correlated with the Taft σ I and σ°R constants; the resonance term was excluded as insignificant.

The positive values of ρ + , ρ I and ρ + R calculated using the δ( 17 O) values for carbonyl oxygen and single-bonded phenoxy oxygen in phenyl esters of substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , (Table 2) prove that the electron-withdrawing substituents cause highfrequency shift of the 17 O signal showing deshielding effect of the O atom. The electrondonating substituents have an opposite effect resulting in low-frequency shift and shielding of the O atom. This is consistent with the increased double bond character of the carbonyl group produced by an electron-withdrawing groups and an increase in single bond character resulting from the electron-donating substituents. [26,36,63,65] An increase in the negative charge on the carbonyl oxygen indirectly causes the oxygen lone pair to be less tightly bound. Earlier [8,9] the increased double bond character of the carbonyl group produced by an electron-withdrawing groups in phenyl esters of substituted benzoic acids was proved by the carbonyl carbon 13 [12] for ortho-substituted methyl benzoates (∆δ

( 17 O) OCH3 = -1.2, ∆δ( 17 O) Cl = 2.9, ∆δ( 17 O) NO2 = -1.7).
The δ( 17 O) values for phenyl esters of ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , listed in Table 1 showed excellent correlations with the Charton equation (Eqns ( 6) and ( 7)) in case the data treatment was carried out separately for derivatives with +R substituents and with -R substituents. In the case of +R substituents (X = H, 2-F, 2-Cl, 2-Br, 2-CH 3 , 2-OCH 3 , 2-N(CH 3 ) 2 ) the influence of ortho substituents to the carbonyl oxygen chemical shifts could be expressed as follows:

(δ 17 O) ortho = (346. 16) was obtained in case the more significantly deviating (δ 17 O) values for 2-NH 2 and 2-I derivatives were excluded before data treatment. In the case of 2-NH 2 ester the deviation was attributed to the intramolecular hydrogen-bonding shielding effect. [9,17] Thus, in the ortho-substituted esters the carbonyl oxygen is deshielded by -I and steric effects and shielded by +R substituent effects.

For phenyl esters of ortho-substituted benzoic acids containing electron-withdrawing -R substituents (X = H, NO 2 , CN, CF 3 , SO 2 CH 3 , SCF 3 ) we found:

(δ 17 O) ortho = (346.9 ± 3.1) + (16.0 ± 5.1)σ I -(32.6 ± 4.0)E s B (17) R = 0.979, s = 2.40, n/n 0 = 6/6
The data treatment with Eqns ( 6) and ( 7) using two steric scales for ortho substituents, E s B and the modified Charton υ constants, gave nearly identical results (Table 2). When the Charton steric υ constants were used, the susceptibility to the steric effect, δ ortho , was positive and approximately twice smaller as compared to the δ ortho value obtained using the E s B scale. We found the influence of ortho substituents to the δ( 17 O) values in phenyl benzoates containing substituents in benzoyl moiety, to be influenced by the positive inductive effect approximately by the same extent for both +R substituents ((ρ I ) ortho = 17.3, Eqn ( 16)) and -R substituents ((ρ I ) ortho = 16.0, Eqn (17)). The influence of the ortho inductive effect was found to be approximately one-half the size of the corresponding influence from the para position (Eqns ( 14) and ( 15)). For phenyl esters of substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , nearly the same ratio (ρ I ) ortho /(ρ I ) para ≈ 2 was observed in the carbonyl carbon 13 C NMR chemical shifts, δ CO , [8] and infrared stretching frequencies, ν CO . [9] For the alkaline hydrolysis of substituted phenyl benzoates in water for both series containing the substituents in benzoyl and in the phenyl part of benzoates (ρ I ) ortho /(ρ I ) para was approximately 1.5. [6] In the case of ortho derivatives with +R substituents the contribution of the throughconjugation between substituent and the carbonyl oxygen was observed by the same extent as that for para substituents. The sensitivity of the δ( 17 O) toward the through-conjugation in the case of ortho +R substituents was 9.68 (Eqn (16), Table 3). The same value for parasubstituted derivatives was 9.71 (Eqn ( 16)). In the case of esters with -R substituents the resonance factor was excluded as insignificant.

Results of correlations in Eqns ( 16) and (17), and Table 2 show that due to the steric positive requirements of ortho substituents the δ( 17 O) values increase (high-frequency shift of the 17 O signal) showing deshielding effect of the O atom in the case of both the ortho +R substituents and ortho -R substituents. We found that the steric requirement in the case of ortho +R substituents was approximately twice as strong as in the case of -R substituents.

In the case of the single-bonded phenoxy oxygen, similar to the inductive and resonance terms, the influence of the steric term appeared to be one-half the size of the steric term for carbonyl oxygen (Table 2). The influence of ortho substituents to the single-bonded δ( 17 O) values in the case of +R substituents could be expressed as follows: with donor +R substituents (Eqn ( 16)) show that the influence of the through-conjugation between ortho +R substituents and the carbonyl oxygen occurs by the same extent as that for para substituents. This confirms that in esters considered with +R substituents the carbonyl group is still coplanar with the ortho-substituted aryl ring and due to the throughresonance effect of electron-donating substituents the carbonyl oxygen shows a strong shielding effect. Consequently, in the ortho-substituted esters with +R substituents only one substituent in the ortho position could not distort the planarity between the carbonyl group and the ring plane essentially. Similarly to esters considered, in the case of the δ( 17 O) values for nitrobenzenes [34] the resonance effect remains important despite of the steric interactions between the substituent and the nitro group. It was concluded [43,65,66] that a small steric effect of ortho substituent in the case of 2-methylbenzoate doesn't necessarily distort planarity or result in steric inhibition of resonance. In a series of substituted benzoate anions, the ab initio calculations of the carbonyl group rotation barriers indicated [67] that due to π-resonance effect the planar conformations are preferred.

We suggest that in the case of +R substituents the observed steric effects in the δ( 17 O) values similar to that for carbonyl carbon 13 C NMR chemical shifts, δ CO , [8] are caused by the repulsive van der Waals deshielding effect. Due to the bulky ortho substituents the πelectron density around the CO oxygen in an ortho-substituted ester would be reduced due to the electrostatic repulsion between the orbitals of the substituent and those of the CO bond, with the effect increasing as substituent increases the size (the van der Waals deshielding [17,41,42,44,68] ). In the case of the electron-donating substituents due to the repulsive van der Waals steric effect the shielding effect of the O atom is reduced.

We found that in the case of the -R electron-withdrawing substituents (X= H, NO 2 , CN, CF 3 , SO 2 CH 3 , SCF 3 ) the steric deshielding effect to be twice weaker as compared to that for the electron-donating substituents and the resonance factor was insignificant. We admit that in the case of -R substituents the carbonyl group is rotated out of the ortho-substituted aryl plane and similar to that for electron-donating substituents, the steric effect is caused by the repulsive van der Waals deshielding effect. [17,41,42,44,68] Evidently, in the case of -R substituents the observed repulsive van der Waals deshielding effect was approximately twice weaker as compared to that for planar esters with +R substituents due to the increased torsion angle between the carbonyl group and the ortho-substituted aryl plane. [13] In the case of ortho electron-withdrawing substituents the deviation from a coplanar configuration between the ring and the carbonyl group is presumably due to prevention of the positive charge delocalization. [36] In phenyl esters of ortho-substituted benzoic acids the calculated sensitivities of the δ( 17 O) values towards the substituent inductive effect, (ρ I ) ortho , was more than three times stronger ((ρ I ) ortho ≈ 17, Table 2) as compared to the same sensitivities, (ρ I ) ortho , in the case of the carbonyl carbon 13 C NMR chemical shifts, δ CO , ((ρ I ) ortho = 5.00 [8] ). In the substituentinduced chemical shifts of carbonyl carbon for phenyl esters of ortho-substituted benzoic 2) was ca 18 times stronger compared to the steric effect in the case of the carbonyl carbon 13 C NMR chemical shifts, δ CO (δ ortho = -4.40). Dahn and Carrupt [26] have reported that the (de)shielding effect of the carbonyl oxygen atom is mainly determined by the energy of the n-π * excitation; donor-acceptor type interactions influence the level of both orbitals. As the n orbital of this transition is nearly exclusively located on the O atom, the extinction acts less on 13 C and thus mainly responsible for the difference in the substituent sensitivity between 13 C and 17 O shifts. Chao [40] concluded that the 17 O shift values in carbonyl compounds are strongly influenced by the n-π * mixing, whereas the 13 C values are determined by multiple factors. The n-π * mixing does not affect the 13 C chemical shifts because the lone pair is located on the O atom. Studying the 17 O chemical shifts in benzamides derivatives De Rosa [69] suggested that in localized exited state the throughconjugation is possible between the substituent and the carbonyl oxygen and is not affected by the ground state torsion angle. We can see (Eqns ( 21) and ( 22), Table 2) that in substituted nitrobenzenes the inductive, resonance and steric substituent effects appeared to be 1.7 times stronger as compared to that for the phenyl esters substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 .

Correlation of the

In the case of ortho-substituted benzoyl chlorides, X-C 6 H 4 COCl, and acetophenones, X-C 6 H 4 COCH 3 , the correlations with the Charton equation (Eqn ( 6)) was performed for derivatives with +R ortho substituents only. Unfortunately in literature there are available only very few values on δ( 17 O) for ortho-substituted benzoyl chlorides and acetophenones with -R substituents, to treat the corresponding δ( 17 O) values with Eqn (6).

For ortho-substituted benzoyl chlorides, X-C 6 H 4 COCl, containing +R substituents we found (Table 2):

(δ 17 O) ortho = (486.0 ± 2.2) + (35.0 ± 2.9)σ I + (16.3 ± 2.5)σ + R -(93.3 ± 4.9)E s B (23) R = 0.994, s = 1.38, n/n 0 = 6/6
In the case of ortho-substituted acetophenones, X-C 6 H 4 COCH 3 , containing +R substituents, the substituent effect is described as follows (Table 2) The δ( 17 O) values for para-substituted nitrobenzenes, benzoyl chlorides and acetophenones showed excellent correlation with Eqn (5) an (6) using the Brown and Okamoto substituent constants σ + . [55,56] The calculated sensitivities towards the through- resonance, ρ + , for para-substituted benzoyl chlorides and acetophenones were respectively 21.2, and 24.1 (Table 2). The corresponding values of ρ + coincide well with the similar ρ + values reported earlier (for benzoyl chlorides ρ + = 21.7, [39] for acetophenones ρ + = 22.5 [27,59] ). For para-substituted nitrobenzenes we obtained ρ + = 14.9 (Table 2, ρ I = 18.6 [34] and ρ + R = 24.0 [34] ). 4)-( 6) as well. The δ( 17 O) values for metasubstituted methyl benzoates showed good correlations so with the single parameter treatment using σ m constants (Eqn ( 4)) as the dual substituent parameter treatment with the Taft σ I and σ°R constants (Table 2). In methyl benzoates for the δ( 17 O) values of the carbonyl oxygen the calculated ρ m value was 5.53 (ρ I = 6.86, ρ°R = 1.93) and for nitrobenzenes ρ m = 5.28 (ρ I = 6.78, ρ°R = 0). The calculated values of ρ m for methyl benzoates and nitrobenzenes coincide well with the similar ρ m values reported earlier (for methyl benzoates ρ m = 6.44 [15] , for nitrobenzenes ρ I = 6.5 [58] , ρ R -= 0.6 [58] . In metasubstituted nitrobenzenes and acetophenones the susceptibility to the inductive effect was ca one-half the size of that for the corresponding para derivatives and the resonance term was excluded as insignificant (Table 2). We correlated the δ( 17 O) values of the carbonyl oxygen and the single-bonded oxygen for phenyl esters of ortho-, para, and meta-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , with the corresponding rates of the alkaline hydrolysis, ∆log k, the carbonyl carbon 13 C NMR chemical shifts, ∆δ CO , and IR carbonyl stretching frequencies, ∆ν CO , using Eqns ( 8)-( 13)

Correlation of the

Correlation of the

(Table 3). The δ( 17 O) values for ortho derivatives, show a good correlation with the corresponding ∆log k values of the alkaline hydrolysis, the carbonyl carbon 13 C NMR chemical shifts, ∆δ CO , and IR carbonyl stretching frequencies, ∆ν CO , (0.998 > R > 0.963, Table 3) in case the additional resonance and steric terms were included (Eqns (8)-( 13)) and the data treatment was carried out separately for esters with +R substituents and -R substituents. The δ( 17 O) values for para derivatives were correlated well with the corresponding values of ∆log k for the alkaline hydrolysis, the 13 C NMR chemical shifts, ∆δ CO , and IR the stretching frequencies, ∆ν CO , when the additional resonance term, a 2(para) σ + R , (Eqns ( 11)-( 13), Table 3) was included. To correlate the δ( 17 O) values for meta derivatives the additional a 2(meta) σ°R scale was included. The slope a 1 in Eqn ( 8)-( 13) is the ratio of inductive effects in the 17 O NMR chemical shifts and in the corresponding process compared. So the slope a 1 in Eqn (8) represents the ratio of the substituent-dependent inductive effects in the δ( 17 O) values and in ∆log k values of the alkaline hydrolysis and the slope a 1 in Eqn ( 9) is the ratio of the susceptibility to the inductive effect in the δ( 17 O) values and in the carbonyl carbon 13 C NMR chemical shifts, ∆δ CO . The slope a 1 in Eqn (10) corresponds to the ratio of the inductive effects in δ( 17 O) values and in the IR stretching frequencies, ∆ν CO , for the ortho-substituted esters. Correlation equations obtained in Table 3 could be used to estimate the rates of the alkaline hydrolysis, the carbonyl carbon 13 C NMR chemical shifts, ∆δ CO , and IR carbonyl stretching frequencies, ∆ν CO , using the δ( 17 O) values for esters considered.

We found the carbonyl 17 O NMR chemical shifts, δ( 17 O), for the phenyl esters of orthosubstituted benzoic acids with +R substituents to be correlated well with the ∆log k values In Eqn (8) a 1 = ρ I ( 17 O)/ρ I (AH), a 2 = ρ R (δ 17 O)a 1 ρ R (AH) and a 3 = δ S (δ 17 O)a 1 δ S (AH). In Eqn (9) a 1 = ρ I ( 17 O)/ρ I (δ CO ), a 2 = ρ R (δ 17 O) -a 1 ρ R (δ CO ) and a 3 = δ S (δ 17 O) -a 1 δ S (δ CO ). In Eqn (10)

a 1 = ρ I (δ( 17 O))/ρ I (ν CO ), a 2 = ρ R (δ 17 O) -a 1 ρ R (ν CO ) and a 3 = δ S (δ 17 O) -a 1 δ S (ν CO ).
In relations shown the alkaline hydrolysis is denoted by (AH), the susceptibility to the steric factor by δ S , the carbonyl carbon 13 C chemical shifts by (δ CO ), and the IR carbonyl stretching frequencies by (ν CO ). Using for the alkaline hydrolysis of the phenyl esters of substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , ρ I (AH) ortho = 2.13, ρ R (AH) ortho = 0.31 and δ(AH) ortho = 2.67 in water at 25 °C [6] as well as ρ I ( 17 O) ortho = 17.3, ρ R ( 17 O) ortho = 9.68 and, δ S ( 17 O) ortho = -81.2 for esters with +R substituents (Table 3) we obtained the values of a 1 = 8.12, a 2 = 7.28 and a 3 = -102.8 which are approximately the same as shown by Eqn (25) (Table 3).

The positive values of the parameter a 1 in Table 3 prove that the substituent-induced δ( 17 O) values, the ∆log k values of the alkaline hydrolysis and the IR carbonyl stretching frequencies, (ν CO ), grow with increase of the inductive effects of substituent included. In correlation of the δ( 17 O) values with the carbonyl carbon 13 C NMR chemical shifts, ∆δ CO , (Eqn (9)) the negative value of the parameter a 1 shows that in phenyl esters of substituted benzoic acids the influence of the substituent-induced inductive effect on the δ( 17 O) values is opposite to that in the 13 When the carbonyl δ( 17 O) values for the phenyl esters of ortho-substituted benzoic acids with -R substituents were correlated with Eqns ( 8)-( 10) (Table 3) the magnitudes of a 2 do not differ from zero and the corresponding resonance term during the data treatment was excluded as insignificant. In the case of esters with -R substituents the influence of the steric term was approximately twice weaker as compared to that for derivatives with +R substituents (Table 3).

When the single bonded δ( 17 O) values were correlated with Eqns ( 8)-( 13) the magnitudes of parameters a 1 , a 2 and a 3 were twice smaller as compared to that for the carbonyl δ( 17 O) values (Table 3).

CONCLUSIONS

The 17 O NMR spectra for 29 phenyl esters of ortho-, para-, and meta-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , were recorded. The electron-donating substituents in ortho-, paraand meta-substituted esters resulted in shielding of the 17 O signal (upfield shift) and electron-withdrawing groups caused deshielding (downfield shift). The 17 O NMR chemical shifts of the carbonyl oxygen and the single-bonded oxygen for para derivatives correlated well with the σ + constants. The δ( 17 O) values for the ortho derivatives showed excellent correlation with the inductive, σ I , resonance, σ + R , and steric, E S B , substituent constants in case the data treatment was performed separately for derivatives containing the electrondonating +R substituents and electron-attracting -R substituents. The steric interaction of ortho substituents with the ester group was found to produce deshielding effect of the carbonyl and single-bonded oxygens. We suggest that in phenyl esters of ortho-substituted benzoic acids the steric effect is caused by the repulsive van der Waals deshielding effect. For ortho derivatives with -R substituents the resonance term was insignificant and the steric term was ca. twice weaker as compared to that for derivatives with +R substituents.

The δ( 17 O) values for para-and ortho-substituted nitrobenzenes correlated well with the σ + , inductive, σ I , resonance, σ + R , and steric, E S B , substituent constants, respectively, similar to that for phenyl esters of substituted benzoic acids. Only, in δ( 17 O), for nitrobenzenes (XC 6 H 4 NO 2 ) the susceptibility to the σ + , inductive σ I , resonance, σ + R , and steric, E s B , substituent constants, was 1.7 times stronger as compared to that for phenyl esters of substituted benzoic acids. The δ( 17 O) values for meta-derivatives correlated well with the σ m substituent constants. [2] . Charton's modified steric constants, υ, [6,8,49] were used. , (R = 0.976, n = 10) using steric constant, υ = 1.22 [60] for OC(CH 3 ) 3 substituent. h For 2-SO 2 CH 3 substituent the steric constant E s B = -0.667 was calculated from relation [6] ∆log k = 0.021 + 2.284σ I + 0.862σ 0 R + 2.477E s B , using ∆log k = -0.309 for alkaline hydrolysis of phenyl ester of 2-(methylsulfonyl)benzoic acid in aqueous 50% DMSO. [51] i Data from References [34,35] were used. j The 2-SCH 3 derivative (E s B = -0.366 was calculated using ∆log k = -0.522 (Note h)), was excluded at confidence level t = 0.99. k For 2,6-(CH 3 ) 2 derivative the duple values of inductive, resonance and the steric constants were used. l For 2-CO 2 CH 3 substituent the isosteric constant E s B = -0.485 was calculated using υ = 1.02 [61] for CH(CH 3 )CH 2 CH 3 substituent (Note g) and for 2-CONH 2 substituent the steric constants for 2-CH(CH 3 ) 2 substituent E s B = -0.341 was used.

F

F

m Data from Reference [39] were used. n Data from Reference. [40] o Data from Reference. [36] p The δ( 17 O) = 604 for 2,6-(CH 3 ) 2 -acetophenone was calculated using ∆δ X value (∆δ X = δ Xδ H = 54). [38] q Data from Reference. [15] r Data from Reference. [14] s Data from Reference [16] t Data from References. [17,57,58] u Data from Reference. [27] v Data from Reference. [59] w Data from Reference. [37] x The δ( 17 O) values for 3-NO 2 , 3-CH 3 and 3-F derivatives were omitted. When these values were included no correlation was found (R 2 = -0.064). The values of ∆log k 1 = log k X -log k H for the alkaline hydrolysis in water at 25 ºC. [6] f

The ∆log k 2 values for alkaline hydrolysis in aqueous 50% DMSO at 25 ºC. [51,52] g

The ∆δ CO values from Reference. [8] h The ν CO values from Reference. [9] i The 2-I and 2-NH 2 derivatives were omitted. j The log k = -1.422 for 2-N(CH 3 ) 2 derivative was calculated from relation: [6] log k = -0.333 + 2.128σ I + 0.312σ 0 R + 2.699E s B .

k The ∆δ CO = 1.1 for 2-N(CH 3 ) 2 derivative was calculated using δ CO from Table 1.

l

The value of ν CO for 2-N(CH 3 ) 2 derivative is not available.

The log k = -0.98 for 2-SCF 3 derivative and the log k = -0.82 for 2-SO 2 CH 3 derivative for alkaline hydrolysis in water were calculated (see Note j). n The ∆log k = -0.566 for 2-SCF 3 derivative and the ∆log k = 1.344 for 2-CN derivative for alkaline hydrolysis in aqueous 50% DMSO were calculated from relation: [6] ∆log k = 0.021 + 2.31σ I + 0.777σ 0 R + 2.693E s B .

o

The ∆δ CO = -0.43 for 2-SCF 3 derivative and ∆δ CO = 0.58 for 2-SO 2 CH 3 were calculated using δ CO (Table 1). p The ν CO values for 2-SCF 3 and 2-SO 2 CH 3 derivatives are not available.

q The log k = -0.055 for 4-Br derivative and the log k = -0.795 for 4-C(CH 3 ) 3 derivative were calculated from relation: [6] log k = -0.451 + 1.72σ.

The ∆log k = -0.464 for 4-C(CH 3 ) 3 derivative for alkaline hydrolysis in aqueous 50 % DMSO was calculated from relation: [6] ∆log k = -0.018 + 2.322σ.

The log k = 0.134 for 3-F derivative, log k = 0.615 for 3-CN derivative, log k = -0.313 for 3-OCH 3 derivative, log k = -0.657 for 3-N(NH 3 ) 2 derivative in water were calculated (see Note q).

t The δ CO values from Table 1 and Reference. [8] u The ν CO values for 3-F, 3-CN and 3-OCH3 derivatives are not available.

  chemical shifts, δ( 17 O), in substituted phenyl benzoates, containing substituents in benzoyl moiety (X-C 6 H 4 CO 2 C 6 H 5 ).

  R = 0.992, s 0 = 0.126, n/n 0 = 11/11 (ν CO ) cis = 1742.7 + 16.4σ I -22.6E s B (2) R = 0.993, s 0 = 0.117, n/n 0 = 9/9 (δ CO ) ortho = 165.32 -5.08σ I + 1.58σ°R -4.33E s B (3) R = 0.978, s 0 = 0.257, n/n 0 = 11/11

13 C

 13 NMR: 131.28 (C-1); 133.79 (C-2), 113.53 (C-3), 136.50 (C-4), 129.69 (C-5), 134.11 (C-6), 163.21 (C-7), 150.77 (C-8), 121.47 (C-9,13), 129.67 (C-10,12), 126.35 (C-11)

13 C

 13 NMR: 132.06 (C-1), 3 J F-C = 7.7; 117.07 (C-2), 2 J F-C = 23.3; 162.79 (C-3), 1 J F-C = 247.4; 120.62 (C-4), 2 J F-C = 21.5; 130.27 (C-5), 3 J F-C = 7.9; 125.94 (C-6), 4 J F-C = 3.3; 164.00 (C-7), 4 J F-C = 3.1; 151.02 (C-8); 121.62 (C-9,13); 129.56 (C-10,12); 126.07 (C-11).

  ) as well. The influence of para benzoyl part of esters on the δ( 17 O) of the single-bonded phenoxy oxygen was ca. one-half the size of substituent effects in the δ( 17 O) of carbonyl oxygen (Table

  O) ortho = (187.7 ± 1.1) + (7.12 ± 1.13)σ I + (2.48 ± 0.70)σ + R -(33.5 ± 3.2)E s B (18) R = 0.986, s = 0.648, n/n 0 = 7/7 Separation of the inductive, resonance and steric components of substituent effects in the δ( 17 O) values for phenyl esters of ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 ,

  substituents the contribution of the steric effect (δ ortho = -81.2, Table

(

  δ 17 O) ortho = (573.7 ± 3.5) + (20.2 ± 6.3)σ I -(58.4 ± 6.0)E s B (20) R = 0.981, s = 3.12, n/n 0 = 6/6 The δ( 17 O) values of carbonyl oxygen for the phenyl esters of ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , showed excellent correlation with δ( 17 O) values for ortho-X-C 6 H 4 NO 2 , including simultaneously derivatives with +R and -R substituents. δ( 17 O) 2-X-nitrobenzenes = (572.2 ± 1.7) + (1.63 ± 0.08)∆δ( 17 O) 2-X-benzoates (21) R = 0.986, s = 2.90, n/n 0 = 12/12 δ( 17 O) 4-X-nitrobenzenes = (575.1 ± 1.4) + (1.70 ± 0.24)∆δ( 17 O) 4-X-benzoates (22) R = 0.935, s = 3.73, n/n 0 = 8/12

  : (δ 17 O) ortho = (550.6 ± 2.3) + (26.2 ± 2.9)σ I + (40.8 ± 2.7)σ + R -(148.0.3 ± 5.4)E s B (24) R = 0.996, s = 1.68, n/n 0 = 7/7

  hydrolysis in water at 25 °C, the carbonyl carbon13 C NMR chemical shifts, ∆δ CO , and the IR stretching frequencies, ∆ν CO , as follows (Eqns (25)-(27), Table3): δ( 17 O) ortho = (346.2 ± 1.7) + (8.90 ± 0.89)∆log k + (7.93 ± 1.10)σ + R -(98.3 ± 5.0)E s B (25) R = 0.993, s = 0.990, n/n 0 = 7/7 δ( 17 O) ortho = (345.4 ± 2.0) -(3.62 ± 0.43)∆δ CO + (10.0 ± 1.3) σ + R -(95.2 ± 5.8)E s B (26) R = 0.991, s = 1.17, n/n 0 = 7/7 δ( 17 O) ortho = (346.3 ± 1.6) + (0.64 ± 0.08)∆ν CO + (7.76 ± 1.56)σ + R -(75.1 ± 4.8)E s B (27) R = 0.990, s = 0.942, n/n 0 = 6/6 The same relationships for the phenyl esters of ortho-substituted benzoic acids with -R substituents are expressed as follows: δ( 17 O) ortho = (347.0 ± 2.9) + (6.31 ± 2.08)∆log k -(50.3 ± 5.5)E s B (28) R = 0.978, s = 2.47, n/n 0 = 6/6 δ( 17 O) ortho = (346.0 ± 1.1) -(3.97 ± 1.14)∆δ CO -(47.4 ± 5.8)E s B (29) R = 0.996, s = 1.09, n/n 0 = 6/6 δ( 17 O) ortho = (345.4 ± 1.9) + (1.32 ± 0.13)∆ν CO (30) R = 0.987, s = 2.09, n/n 0 = 4/4

gFor 2 -

 2 SCF 3 substituent the isosteric constant E s B = -0.582 was calculated with relation: υ = (0.026 ± 0.049) -(2.05 ± 0.15)E s B

y

  The δ( 17 O) values for 3-NO 2 , 3-CH 3 and 3-F derivatives were omitted. When these values were included no correlation was found (R 2 = 0.054). z The 3-SO 2 NH 2 derivative was excluded. aa The 3-SO 2 NH 2 , 3-SO 2 CH 3 , 3-SO 2 Cl, and 3-CH 2 Cl derivatives were excluded.

F

Table

  ). The 17 O NMR spectra were recorded on a Bruker Avance II 200 spectrometer equipped with 10 mm BBO probe at the resonance frequency 27.13 MHz. Benzoates were dissolved in the mixture of 1 ml CD 3 CN and 2 ml CH 3 CN. Acetonitrile-d 3 "Special HOH" 99.8 atom % D, Aldrich and Acetonitrile SPECTRANAL R , Riedel-de Haёn were used. Depending on the type of the benzoate the concentration varied

	F o
	r
	P
	e e r
	R e v i e w

between values from 0.3 molal to 1 molal. Temperature of the measurements was 50 °C. Water at 50 °C was used as an external reference ( 17 O chemical shift was taken 0 ppm). Depending on the sample about 0.5 to 1.8 million scans (measurement time up to 70 hours)

Table 2 )

 2 In the case of ortho-substituted nitrobenzenes, X-C 6 H 4 NO 2 , with -R substituents (X = H, NO 2 , CN, CF 3 , CONH 2 , CO 2 CH 3 ) the substituent effect (δ 17 O) ortho is expressed as follows (:

	CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , C(CH 3 ) 3, OCH 3 , SCH 3 , N(CH 3 ) 2 , F, Cl, Br, 2,6-(CH 3 ) 2 ) we
	obtained (Table 2):
	(δ 17 O) ortho = (578.1 ± 3.5) + (26.8 ± 5.2)σ I + (11.7 ± 2.4)σ +	R -(108.4 ± 7.8)E s B	(19)
	R = 0.975, s = 3.47, n/n 0 = 11/12

17 O NMR chemical shifts, δ δ δ δ( 17 O), for orthoand para-substituted

nitrobenzenes, benzoyl chlorides and acetophenones

To compare the substituent effects in the δ( 17 O) values for ortho-and para-substituted nitrobenzenes, X-C 6 H 4 NO 2 , benzoyl chlorides, X-C 6 H 4 COCl, and acetophenones, X-C 6 H 4 COCH 3 , with those in δ( 17 O) values for the phenyl esters of para-and orthosubstituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , the corresponding δ( 17 O) values were correlated with Eqns (4)-

(6)

.

Similarly to the phenyl esters of ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , (Eqns (

16

) and (

17

)), the δ( 17 O) values for ortho-substituted nitrobenzenes showed excellent correlation with the Charton equation (

6

) in case the data treatment was performed separately for nitrobenzenes with +R substituents and with -R substituents. For δ( 17 O) values of ortho-substituted nitrobenzenes, X-C 6 H 4 NO 2 , with +R substituents (X = H,

for meta-substituted methyl benzoates, nitrobenzenes, and acetophenones

	C 6 H 4 COCH 3, were correlated with Eqns (
	17 O NMR chemical shifts, δ δ δ o r P e e r δ( 17 O), F R e v i e w
	14

For comparison the δ( 17 O) values published in literature for meta-substituted methyl benzoates, X-C 6 H 4 COCH 3 , nitrobenzenes, X-C 6 H 4 NO 2 , and acetophenones, X-

  C NMR chemical shifts, ∆δ CO . With increase in σ I values of substituents the δ( 17 O) values were found to increase but the carbonyl carbon13 C NMR ∆δ CO , were found to diminish when the electron-attracting substituents are involved.

	chemical shifts,
	F o
	r
	P
	e e r
	R e v i e w
	16

Table 1 .

 1 17 O NMR chemical shifts, δ( 17 O), of the carbonyl oxygen and the single-bonded oxygen for phenyl esters of ortho-, para-and metasubstituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5 , in CH 3 CN at 50 °C Approximately 1 molal solutions of ortho and para derivatives in mixture of CH 3 CN and CD 3 CN (2 ml + 1 ml) were used, except where shown otherwise. b Reference

	o
	r
	P
	e e r
	R e v i e w

a

Table 2 .

 2 Correlation of the 17 O NMR chemical shifts, δ( 17 O), of the carbonyl oxygen and the single-bonded oxygen (OPh) with Eqns (4)-(7) n 0 reflects the total number of data involved in the correlation; n -the number of points remaining after exclusion of significantly deviating points.The δ( 17 O) values for 2-NH 2 and 2-I derivatives were omitted.

	Scales	δ( 17 Ο) H ρ Ι or ρ + (ρ) ρ +	R (ρ°R)	δ ortho	R a (R 0	2 ) b s c	n/n 0 d
				Phenyl esters of ortho-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5
				C= 17 O	+R substituents included, acetonitrile at 50 °C
	σ I , σ +	R , E s B 346.2 ± 1.8 17.3 ± 1.8 9.68 ± 1.13 -81.2 ± 5.2 0.993 0.182 1.06	7/7 e
	σ I , σ +	R , υ 347.4 ± 3.5 14.3 ± 4.3 4.62 ± 2.08 33.2 ± 5.0 0.961 0.182 2.41	7/7 e,f
					-R substituents included, acetonitrile at 50 °C
	σ I , σ + σ I , υ	o B 346.9 ± 3.1 16.0 ± 5.1 R , E s 346.5 ± 2.0 18.1 ± 1.3 r 17 OC 6 H 5 +R substituents included, acetonitrile at 50 °C 0 -32.6 ± 4.0 0.979 0.304 2.40 0 15.9 ± 1.3 0.991 0.304 1.61	6/6 g 6/6 f
	σ I , σ + σ I , σ +	R , E s B 187.7 ± 1.1 7.12 ± 1.13 2.48 ± 0.70 -33.5 ± 3.2 0.986 0.100 0.648 P R , υ 188.2 ± 1.1 6.04 ± 1.85 0 13.0 ± 1.7 0.963 0.100 1.06	7/7 e 7/7 e,f
	σ I , E s B σ I , υ	-R substituents included, acetonitrile 50 °C e 187.3 ± 1.2 5.52 ± 1.90 --15.5 ± 1.5 0.986 0.225 0.895 6/6 g,h e 187.1 ± 1.7 6.76 ± 2.80 -7.35 ± 1.10 0.967 0.225 1.35 6/6 f r ortho-substituted nitrobenzenes, X-C 6 H 4 NO 2
	N( 17 O) 2 B 578.1 ± 3.5 26.8 ± 5.2 11.7 ± 2.4 -108.4 ± 7.8 0.975 -0.072 3.47 11/12 e,i,j,k +R substituents included, acetonitrile at 75 °C R , E s R -R substituents included, acetonitrile e B σ I , σ + 573.7 ± 3.5 20.2 ± 6.3 0 -58.4 ± 6.0 0.981 0.072 3.12 6/6 l σ I , E s ortho-substituted benzoyl chlorides, X-C 6 H 4 COCl v C= 17 O +R substituents included, acetonitrile at 75 °C i σ I , σ + R , E s B 486.0 ± 2.2 35.0 ± 2.9 16.3 ± 2.5 -93.3 ± 4.9 0.994 -0.188 1.38 6/6 k,m,n e ortho-substituted acetophenones, X-C 6 H 4 COCH 3 C= 17 O +R substituents included, dioxane at 30 °C w σ I , σ + R , E s B 550.6 ± 2.3 26.2 ± 2.9 40.8 ± 2.7 -148.0 ± 5.4 0.996 -0.187 1.68 7/7 k,o,p
				Phenyl esters of para-substituted benzoic acids, X-C 6 H 4 CO 2 C 6 H 5
				C= 17 O	Acetonitrile at 50 °C
	σ +	p		345.2 ± 0.3 9.07 ± 0.55	-	-	0.986	-0.918	9/9
	σ I , σ +	R	345.9 ± 0.5 7.21 ± 0.99 9.71 ± 0.54	-	0.990 0.106 0.752	9/9
				17 OC 6 H 5
	σ +	p		187.1 ± 0.2 2.26 ± 0.26	-	-	0.952	-0.428	9/9
	σ I , σ +	R	187.5 ± 0.2 1.26 ± 0.41 2.60 ± 0.22	-	0.976 0.004 0.307	9/9
				Methyl esters of para-substituted benzoic acids, X-C 6 H 4 CO 2 CH 3
				C= 17 O	Acetonitrile at 40 °C
	σ +	p		337.8 ± 0.2 9.66 ± 0.29	-	-	0.990	-0.970 23/23 q
						22

a R -correlation coefficient. b R 0 -zeroth correlation coefficient. c s -standard deviation. d e f

  n 0 reflects the total number of data involved in the correlation; n -the number of points remaining after exclusion of significantly deviating points.

	∆log k 1 , σ°R ∆δ CO , σ°R ∆ν CO , σ°R	346.0 ± 0.2 4.22 ± 0.37 346.1 ± 0.2 -2.20 ± 0.16 346.0 ± 0.9 0.61 ± 0.08	---	---	0.974 -0.521 8/8 s 0.981 -0.447 8/8 t 0.970 -0.624 5/5 u
	a R -correlation coefficient.
		o F o
		r r
		P P
		e e r e e r
		R e v i e w R e v i e w
		27

b R 0 -zeroth correlation coefficient. c s -standard deviation. d e

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Acknowledgements

This work was supported by the grants No 8162 and No 6705 of the Estonian Science Foundation.


C Sheridan
email: c.m.sheridan@liv.ac.uk

Expanded Polytetrafluoroethylene as a Substrate for Retinal Pigment Epithelial Cell Growth and Transplantation in Age-related Macular Degeneration

Background: Retinal pigment epithelial (RPE) transplantation presents a potential treatment of Age-related Macular Degeneration (AMD). A suitable transplant membrane that can support an intact functioning RPE monolayer is required. Expanded polytetrafluoroethylene (ePTFE) possesses the physical properties required for a transplanting device, however, cells do not attach and spread on ePTFE. This study investigated the ability of surface modified ePTFE to optimise the growth and function of healthy RPE monolayers. Methods: ePTFE discs were modified by ammonia gas plasma treatment. ARPE-19 cells were seeded on the membranes and maintained in media supplemented with retinoic acid and reduced serum. Cell number, morphology and proliferation were analysed. RPE monolayer function was investigated through formation of cell-cell junctions and phagocytosis of photoreceptor outer segments (POS).

Results: Ammonia gas plasma treatment resulted in enhanced cell growth, good monolayer formation with evidence of cell-cell junctional proteins. Furthermore, RPE monolayers were able to phagocytose POS in a time-dependent manner.

Conclusions: ePTFE can be surface modified to support an intact functional monolayer of healthy RPE cells with normal morphology and the ability to perform RPE-specific functions. With further investigation ePTFE may be considered for use in transplantation.

Introduction

Replacing retinal pigment epithelial cells under the failing macula represents a potential treatment for age-related macular degeneration. [START_REF] Coffey | Long-term preservation of cortically dependent visual function in RCS rats by transplantation[END_REF][START_REF] Van Meurs | Autologous peripheral retinal pigment epithelium translocation in patients with subfoveal neovascular membranes[END_REF][START_REF] Wong | Case selection in macular relocation surgery for age related macular degeneration[END_REF][START_REF] Sheridan | Replacement of the RPE monolayer[END_REF] For transplants to be successful they will need to be introduced under the macula as an intact functioning monolayer as delivery of isolated cells can cause serious complications including PVR. [START_REF] Hiscott | Matrix and the retinal pigment epithelium in proliferative retinal disease[END_REF] A range of biological substrates and degradable synthetic substrates have been investigated but are of limited use due to lack of reproducibility of the biological substrates or the potential to leave cells exposed to underlying damaged Bruch's membrane following degradation, as well as the unknown fate of the degradation products. 4;6-8 The aim in this study was to identify if expanded polytetrafluoroethylene (ePTFE) is a suitable biostable substrate and address the options to enhance its properties required for transplantation. ePTFE is a fluoropolymer, which has been used in a range of surgical devices with well-established biocompatibility, biostability, porosity and flexibility. 9;10 ePTFE 'as received,' however, is hydrophobic and does not promote cell adhesion.

Previous studies have shown that surface modification can enhance cell attachment. 9;11 The present study investigated how surface modification of ePFTE using an ammonia gas plasma treatment enhanced the growth of RPE cell monolayers in the presence of retinoic acid and reduced serum levels and furthermore preserved their differentiation and functional characteristics.

Materials and Methods

Substrate and plasma treatment

Samples 3mm in diameter were punched from a 100μm thick ePTFE sheet and cleaned in 100% ethanol. A scanning electron micrograph of the ePTFE is presented in Figure 1. Untreated samples were placed into sterile distilled water and stored until cell culture experiments. The remaining discs were surface modified by ammonia gas (100% purity; BOC Ltd, Surrey, UK) plasma treatment, following the procedure previously reported. 12;13 Following treatment, the samples were placed directly into sterile distilled water for 24 hours prior to use in cell culture. No change in the surface topography was observed after plasma treated under scanning electron microscopy. All samples were UV sterilised (CL-1000 U.V. Crosslinker; UVP, Cambridge, UK) for 3 minutes immediately before use in cell culture.

Cell culture

ARPE-19 cells, an established but non-immortalised human RPE cell line (American

Type Culture Collection, Manassas, VA, USA), were seeded on ammonia gas plasma treated ePTFE samples and controls (tissue culture polystyrene; TCPS) at a density of 1500 cells/mm 2 . The samples were maintained in a 1:1 (vol/vol) mixture of DMEM/F12 (Sigma-Aldrich Ltd, Ayrshire, UK) containing 2mM L-glutamine (Gibco, Paisley, UK), 50U/ml penicillin G, 50μg/ml streptomycin (Gibco), 2.5ug/ml amphotericin B (Gibco), and supplemented with 10% FBS (Gibco) for the first 4 days and incubated at 37 o C with 5% CO 2 . From day 4 cells were grown in culture media with 2.5% serum levels supplemented with 10μM all-trans retinoic acid (RA) (Sigma), as described in previous studies. [START_REF] Dunn | ARPE-19, a human retinal pigment epithelial cell line with differentiated properties[END_REF][START_REF] Campochiaro | Retinoic acid promotes densitydependent growth arrest in human retinal pigment epithelial cells[END_REF][START_REF] Yasunari | Effect of retinoic acid on proliferation and polyamine metabolism in cultured bovine retinal pigment epithelial cells[END_REF] At 1, 4, 7, 10 and 15 day time points the samples were fixed in 10% neutral buffered formalin. Primary human RPE cells (hRPE) were isolated and expanded as described in Kent et al., 2003 [START_REF] Kent | Edible mushroom (Agaricus bisporus) lectin inhibits human retinal pigment epithelial cell proliferation in vitro[END_REF] and seeded at a density of 800 cells/mm 2 (cells were used from 2 donor eyes and were passage number 5-12). Samples were maintained F10 medium (Sigma) containing 2mM L-glutamine, 50U/ml penicillin G, 50μg/ml streptomycin, 2.5ug/ml amphotericin B, and supplemented with 20% FBS. At day 2, FBS was reduced to 5% and medium was supplemented with 10μM RA.

Samples were fixed at 7, 14, 21 and 28d.

Quantification of cell number (nuclear counts)

ARPE-19 cells were nuclear stained with 10μg/ml propidium iodide (Molecular Probes, UK) and visualised using fluorescence microscopy. Five fields were photographed per substrate (three replicate substrates per time period). The mean number of nuclei per field of view (2.76x10 -2 mm 2 ) was calculated for each time interval for each treatment and control.

Qualitative assessment of cellular cytoskeletal morphology

Fixed samples were stained with 5U/ml Alexa Fluor 488 phalloidin (Molecular Probes) F-actin stain and visualised by fluorescence microscopy.

Identification of proliferating cells

At Day 4, 7 and 15 ARPE-19 cells grown on treated ePTFE and control TCPS surfaces were incubated with a 10μM bromodeoxyuridine (BrdU; Sigma) solution in serum-free media for 2 hours at 37 o C. Samples were fixed and stained with 10μg/ml murine monoclonal anti-BrdU Alexa Fluor® 488 conjugate antibody (Molecular Probes).

Mouse IgG 1 -(FITC) 10μg/ml was used as a negative control. Samples were counterstained with 10μg/ml propidium iodide and visualised using fluorescence microscopy. The nuclei of proliferating cells were counted and expressed as a percentage of the total number of cells on the substrate.

Immunohistochemistry for cell-cell junctions

Cell-cell contact and monolayer formation were evaluated by staining for protein components of tight junctions (ZO-1 and occludin), for adherens junctional proteins (cadherins), and for gap junctional protein connexin 43. Treated substrates and control TCPS were fixed and incubated with 10μg/ml murine monoclonal anti-ZO-1-(FITC) (Invitrogen, Paisley, UK). Mouse IgG-(FITC) (Dako) 10μg/ml was used as a negative control. Duplicate samples were incubated with 10 μg/ml rabbit anti-ZO-1, 10μg/ml rabbit anti-pancadherin (Invitrogen), 2μg/ml rabbit anti-occludin (Invitrogen) or 2.5μg/ml rabbit anti-connexin 43 (Sigma) antibodies for 1 hour RT post blocking for 30 minutes with 10% goat serum (Dako). Rabbit IgG (Dako) was used as a negative control.. After 1 hour the samples were rinsed and incubated with 20μg/ml secondary antibody Alexa Fluor 488 or 594 goat anti-rabbit. All samples were visualised using fluorescence microscopy. At least 3 replicate experiments were performed with 6 samples in each experiment.

Phagocytosis of photoreceptor outer segments (POS)

The methods used to harvest and label the POS were adapted from procedures previously described. [START_REF] Miceli | Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium[END_REF];19 ARPE-19 were seeded onto the treated ePTFE and control substrates and incubated as above. All samples were treated with 10μM RA in culture media with 5% FBS at Day 4. At Day 7, when cells were known to have reached confluence, each of the samples was rinsed with fresh media and challenged with 10μg/ml of POS and then incubated for 3 hours and 24 hours. At each time-point, samples were rinsed with PBS, mounted in glycerol at pH 9 and immediately visualised using fluorescence microscopy. Ten fields of view were photographed per substrate per time interval and the number of attached (which fluoresced red at the external pH 9) and internalised POS (which fluoresced green at an acidic pH inside phagosomes) were counted. The mean number of attached and internalised POS per field of view (2.76x10 -2 mm 2 ) were recorded for ePTFE and control for each time-point.

Results

Cell number and morphology

Both ARPE-19 and hRPE demonstrated well-defined circumferential actin staining (Figure 2) with homogenous cell coverage of the ePTFE surface and close cell packing.

The number of ARPE-19 cells (Figure 3) attached to the treated ePTFE was lower than that attached to the control TCPS at each time point but the number was increasing on both surfaces. Very few ARPE-19 cells were counted on untreated ePTFE at all time points.

Identification of proliferating cells

At Day 4 the bright green BrdU staining clearly highlighted a high proportion of nuclei, from the total number of nuclei, on both ePTFE and control TCPS surfaces (68.4% and 71.6%, respectively) (Table 1). At Day 7, however, only a very small percentage of nuclei took up the BrdU stain from the total (0.7% and 1.3%, respectively). By Day 15 the mean percentage of BrdU labelled nuclei further dropped (0.5% and 0.6%, respectively). The staining, however, could only be demonstrated in a small area of cell overgrowth on both surfaces at Day 7 and 15 and was not present in all other areas of the substrate and control, where a homogenous coverage of the surfaces was observed.

Overall, the number of proliferating cells noted on ePTFE was lower than that seen on control (p=0.047) (Table 1).

Immunohistochemistry for cell-cell junctions:

Presence of tight junctional protein components, ZO-1 and occludin, were demonstrated at Day 7 by ARPE-19, on ePTFE and control TCPS (Figures 4a and4b) at cell-cell borders rather than perinuclear. Furthermore, their staining could only be shown in confluent monolayers and not in pre-confluent cells before Day 7. Circumferential pancadherin staining was evident on both surfaces (Figures 4c). The gap junctional protein, connexin 43, was also extensively observed by the bright punctate immunofluorescent staining at cell-cell contacts over the whole surface area on both ePTFE and controls. hRPE exhibited similar trends, although junctional protein components were not observed until later time points. ZO-1 was observed only in small patches at Day 7, but was visible over the whole surface area at 14 and 28 days (Figure 4d). Similar results were obtained when cells were stained for occludin, pancadherin and connexin 43.

Phagocytosis of photoreceptor outer segments (POS)

Cells attached to the ePTFE were able to phagocytose the fluorescently labelled POS in a time-dependent fashion and in a similar manner to the cells on the control TCPS. At 3 hours a mean of 61 (±6.0) POS were attached to the cell membranes and a mean of 12.7 (±8.9) POS were internalised on the ePTFE whereas 65.4 (±9.2) were attached to the cells adhered to the control and 21.2 (±3.6) were internalised (Figure 5). By 24 hours the mean attached and internalised POS were very similar for both ePTFE and the control. There was a significant difference for both attached and internalised POS between 3h and 24h (p<0.001) for the 2 surfaces together. However, there was no statistically significant difference found between control and ePTFE.

Discussion

AMD is a major problem with between 20-25 million people affected worldwide and the figure is expected to triple in the next 40 years with the growing ageing population. [START_REF] Chopdar | Age related macular degeneration[END_REF] For RPE transplantation to be a clinical possibility it is essential to provide a membrane on which to grow the RPE cells that will provide a replacement for the natural Bruch's membrane and maintain the health and functionality of the transplanted cells for the patient's lifetime. The approach was to investigate ePTFE as a biostable synthetic membrane that would satisfy the prerequisites of a suitable transplanting device, and support growth of a healthy intact functioning monolayer of RPE cells.

Previous studies have shown ammonia gas plasma treatments followed by a posttreatment storage in distilled water results in a defluorination of the fluoropolymer surface and the incorporation of surface polar groups. 12;13 This increase in hydrophilicity promotes cell attachment and growth.

Once a monolayer of cells has formed it is important to arrest cells in their differentiated state (G 0 phase). Retinoic acid (RA) performs this function in the eye and is known to promote RPE differentiation during development. [START_REF] Campochiaro | Retinoic acid promotes densitydependent growth arrest in human retinal pigment epithelial cells[END_REF] A differentiated monolayer was achieved on ePTFE in the presence of RA with homogeneous coverage of the substrate surface. Growth arrest and evidence that a differentiated monolayer will remain is vital, prior to implantation into the eye, in order to ensure there is less risk of developing complications such as PVR or neoplasia. This was further confirmed by the very low percentage uptake of BrdU staining, which was used as a marker of cell proliferation, by the RPE cells grown on ePTFE at the later time-points. Furthermore, culture of RPE cells in reduced serum levels would probably give a realistic indication of their survival and maintenance as a monolayer if implanted into the eye, especially in patients with geographic atrophy and associated atrophy of the choriocapillaris.

The ability of RPE cells to polarise and form a monolayer is dependent on the formation of junctional complexes typical for an epithelial cell phenotype. The formation of tight junctions (of which ZO-1 and occludin are protein components) depends on the integrity of the adherens junctions (which contain cadherins). 14;21 Good cell-cell junctional staining was exhibited by both cell types for ZO-1, occludin, pancadherins and gap junctional protein connexin 43 on treated ePTFE. Formation of functional junctional complexes is important for effective barrier function, which is an essential function of RPE cells in vivo.

Failure of RPE to phagocytose POS has been well documented to cause retinal degenerations, including AMD where the macula is particularly predisposed because of its high photoreceptor density. [START_REF] Chaitin | Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells[END_REF][START_REF] Bergmann | Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration[END_REF][START_REF] Nilsson | Aging of cultured retinal pigment epithelial cells: oxidative reactions, lipofuscin formation and blue light damage[END_REF] Hence many studies have targeted their investigation into factors controlling POS attachment and internalisation as a measurement of phagocytosis. 25;26 Cells grown on ePTFE substrates were able to phagocytose POS in time dependent manner similar to that of cells on the control TCPS. Findings from this study, however, were consistent with those reported in earlier studies showing large numbers of isolated POS phagocytosed by confluent RPE cell cultures in vitro. [START_REF] Chaitin | Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells[END_REF] Previous studies have used RPE cells grown on control TCPS as a comparator in phagocytosis assays 18;19 and thus were used as controls for the present study.

Phagocytosis of POS has also been demonstrated by IPE cells, although at a lower rate to that of RPE cells. 19;27 This is important when considering which would be the appropriate cell type to eventually transplant. It would be essential to ascertain whether the candidate cell types such as genetically modified RPE cell-lines, primary RPE or IPE, or RPE derived from stem cells, would still be able to phagocytose POS and maintain visual function and how the subretinal environment may influence this in vivo. 4;28 In summary, this study demonstrated that the surface properties of ePTFE could be manipulated by gas plasma treatment to support the growth of an intact functional monolayer of RPE cells. Furthermore, by controlling the cell culture conditions in terms of addition of differentiating agents and serum reduction, it was possible to optimise and maintain a monolayer of well spread cells displaying evidence of intercellular tight junctions. The cells grown on gas plasma modified ePTFE were able to phagocytose POS in a time dependent manner, consistent with those reported in previous studies, demonstrating an essential function of RPE cells in vivo. The advantages of using a biostable membrane rather than a synthetic biodegradable or tissue-based material will be the long term support and an effective disease-free basement membrane for the transplanted cell monolayer. Thinner ePTFE membranes are available and will be required for future in vivo studies.

Although it is recognised that ARPE-19 cells in their present state would not be used for subretinal transplantation, results from this well-characterised cell-line and the observation of similar trends in terms of cell morphology and cell-cell junction formation using primary human RPE provide compelling evidence that ePTFE may be considered as the membrane of choice to proceed with future in vivo experiments.

Fight for Sight. The authors would like to thank M García-Fiñana for her help with statistical analysis.

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Fig 1 :

 1 Fig 1: Representative scanning electron micrograph of ePFTE.

Fig 2 :

 2 Fig 2: Representative photomicrographs displaying the cell cytoskeletal morphology of

Fig 3 :

 3 Fig 3: Histogram showing the mean cell number of ARPE-19 (nuclear counts) per field

Fig 4 :

 4 Fig 4: Cell-cell junctional staining of ARPE-19 cells at Day 7 grown on ammonia gas

Fig 5 :

 5 Fig 5: Histogram demonstrating mean number of POS per field of view attached and

Acknowledgements

Work for this study was kindly supported by Research & Development Support Fund, Royal Liverpool and Broadgreen University Hospitals, The Dunhill Medical Trust and