15 N Performance Validation Ss


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The authors of this technical note recently authored a chapter entitled “Applications of 15N NMR in Alkaloid Chemistry” for publication in Modern Alkaloids (Editors E. Fattorusso and O. Taglialatela-Scafati) to be published by Wiley in 2007. During the preparation of this article we took advantage of the opportunity to validate the performance of the ACD/NNMR predictor by applying it to the prediction of nitrogen chemical shifts associated with the diverse and challenging structures found in alkaloids. The results of this study are reported here.

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15 N Performance Validation Ss

  1. 1. Technical Note Performance Validation of the ACD/Labs 15 N Shift Prediction Software ACD/NNMR Version 10.0 Dr. Antony J. Williams1 and Dr. Gary E. Martin2 1) Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com 2) Schering-Plough Corporation, Pharmaceutical Sciences, 556 Morris Ave., Summit, NJ 07901 Introduction The authors of this technical note recently authored a chapter entitled “Applications of 15N NMR in Alkaloid Chemistry” for publication in Modern Alkaloids (Editors E. Fattorusso and O. Taglialatela- Scafati) to be published by Wiley in 2007. During the preparation of this article we took advantage of the opportunity to validate the performance of the ACD/NNMR predictor by applying it to the prediction of nitrogen chemical shifts associated with the diverse and challenging structures found in alkaloids. The results of this study are reported here. 15 N Chemical Shifts 15 The N chemical shift range is very broad, encompassing approximately 900 ppm. For natural products, however, the majority of 15N resonances of interest will be in the range from about 20-350 ppm, with the exception of a few functional groups such as nitro groups that resonate in the vicinity of ~ 380 ppm and methoxime (=N-OCH3) groups that resonate at ~405 ppm. 15N Chemical shifts of alkaloids range from those of amino groups, which are generally ~ 20-60 ppm down through thiazolyl and pyridyl nitrogens which resonate in the range of about 300-340 ppm (relative to NH3). 15 Even with the restriction of the N chemical shift range of interest to 20-350 ppm, this spectral window is still experimentally challenging from the standpoint of experiment parameterization. At the low observation frequency of 15N (~60 MHz on a 600 MHz instrument), it is difficult to generate observe pulses short enough to effectively cover a 300+ ppm spectral window. For this reason it is preferable, when an investigator is working with a system that they understand reasonably well, to limit the F1 spectral window to whatever extent possible. The use of 15N chemical shift calculation algorithms such as Advanced Chemistry Development’s ACD/NNMR software can help in this regard.
  2. 2. Technical Note 15 N Chemical Shift Calculation and Prediction 15 Structure verification using a N content database For the chemist attempting to elucidate a chemical structure the 15N chemical shift is a sensitive probe of the nitrogen environment. For the purpose of structure verification a common approach is to review the literature for related species and use their chemical shifts and couplings as models to allow estimates of these properties for the new species. While there are a number of texts, reviews, and publications available that have brought together the spectral properties of tens to hundreds of molecules these paper-based collections are cumbersome to use when it comes to searching for a particular chemical shift or a chemical structure or substructure. With both time and quality of the essence for such searches of data, the most obvious approach is to compile an appropriate collection of data into an electronic database and enable the appropriate types of searches. When a content database of chemical structures and associated spectral parameters is made available, this can greatly speed up the process of identifying the nature of the compound. Electronic content databases are available from a number of sources. The largest and most up to date source of 15N data is that supplied by ACD/Labs. The content database is delivered with their ACD/NNMR Predictor program. It can be searched by structure, substructure, similarity of structure, chemical shift or range of chemical shifts as well as coupling constants. Add to this the ability to search through the databases by formula and mass (nominal, average, or exact) and an NMR spectroscopist has immediate access to a warehouse of valuable information. The ACD/NNMR v10 content database contains > 8800 chemical structures (> 21,000 15 N chemical shifts). These data have been culled from the literature and checked for quality according to a number of stringent criteria prior to adding to the database. The chemical shift reference is homogenized during the process such that all shifts are relative to one reference (even though predictions can be referenced to four common standards: liq. NH3, NH4Cl, HNO3 and CH3NO2). A single database record includes the chemical structure, the original literature reference, the 15N chemical shift(s) and, where available, associated heteronuclear coupling constants. The database is updated on an annual basis with new data extracted from the literature. This database is also the foundation of data supporting the prediction algorithms that are required to predict NMR spectral properties for chemical structures not contained within the database. 15 N NMR prediction NMR prediction brings the possibility of structure verification based on chemical shifts as well as offering the opportunity of using prediction to optimize experimental acquisition parameters and sweep widths for the acquisition of 2D spectra, a valuable facility considering the example of the survey conditions applied to harmaline as discussed earlier. ACD/Labs uses proprietary algorithms based on a modified form of HOSE-code technology. In order to perform a prediction the user simply sketches the chemical structure of interest using a structure editor. The calculation of the chemical shifts and coupling constants is performed in a matter of seconds. A resulting table of chemical shifts displays the number of the atom in the structure that gives rise to the predicted shift; the value of the predicted shift; and the uncertainty of the predicted shift, based on 95% confidence limits for the structure fragment. The table also includes predicted coupling constants between pair of atoms. It is possible to determine how the chemical shifts were predicted, and the type of structural fragments used to derive the parameters though a Calculation Protocol window which shows a series of points, each representing an individual chemical structure and associated 2
  3. 3. Technical Note chemical shift that was used to influence the chemical shift prediction. If the prediction was performed on a compound not in the database, then a variety of different structures are shown in the Calculation Protocol and displayed as a histogram plot containing structures which are only fragmentally similar to the input structure. The general applicability and success of 15N NMR prediction will be examined in further detail below. 15 Validating N NMR prediction 15 The validation of N NMR prediction is best performed by comparing the predicted shifts for compounds not in the database with the experimental shifts available in the literature or measured directly. As described earlier the prediction algorithms are derived from a training set of over 21,000 chemical shifts. The training set is upgraded on an annual basis based on published literature data. For the chemical shifts reported in the chapter prepared for the Modern Alkaloids book almost 75% of the data reported are contained within the database presently associated with the version 10 release. For the remaining 25% of chemical shifts listed in the chapter a regression analysis was performed to compare experimental versus predicted chemical shifts. 2 The results are represented in Figure 1. Regression delivers a value of R = 0.987, an excellent correlation and demonstrative of the performance of the NMR prediction algorithms. Experim ental Versus Predicted N15 Shifts 350 300 250 Predicted Shifts 200 150 100 50 y = 0.9702x R2 = 0.9867 0 0 50 100 150 200 250 300 350 Experim ental Shifts 15 15 Figure 1. A plot of observed vs. calculated N chemical shifts for 49 N chemical shifts from the Modern Alkaloids chapter. These chemical shifts and associated compounds are not contained within the training set. Regression analysis delivered: R2 = 0.987, standard error 14.8 ppm. 3
  4. 4. Technical Note Conclusions 1 1 15 These authors have previously reported on the best practices to follow when acquiring H – N heteronuclear shift correlation data. In addition that report described on the validation of the performance of the ACD/NNMR software for prediction of 15N chemical shifts and 1H –15N heteronuclear coupling constants. In that work is was concluded that 15N shift calculations can make the difference between recording meaningful data and wasting spectrometer time by examining a series of particular structure examples an comparing experimental to predicted data. This technical note has analyzed a larger data set comprised of extremely diverse chemical structures from a family of alkaloids and has demonstrated excellent performance in terms of overall performance. The ACD/NNMR database will continue to grow as new data are culled from the literature and new releases are issued annually. With a larger database and increased 15 structural diversity, further refinement of the prediction algorithms will be facilitated and N chemical shift predictions will continue to improve. References 1. G.E. Martin and A.J. Williams, A Best Practice Guide for Acquiring 1H –15N Heteronuclear Shift Correlation Data, http://www.acdlabs.com/download/technotes/90/nmr/shift_correlation.pdf 4