No part of this document may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into any language in any form by any means without the written permission of Spectrum Research, LLC.

Spectrum Research, LLC. reserves the right to change the information in this document without prior notice.

Trademarks

SpecMan and NMR-SAMS are trademarks of Spectrum Research, LLC.

Acknowledgments

Computer-Assisted Structure Elucidation of Q-2
Using SpecMan and NMR-SAMS

Overview:

The following document provides step-by-step instructions that lead you through the process of computer-assisted structure elucidation of the Q-2 (betulinic acid) molecule. This document is intended for the day-to-day users of SpecMan and NMR-SAMS and we assume the users of this document to have a good understanding of general NMR techniques and its application to structure elucidation. There are two parts to this document. Part I describes the step-by-step instructions that lead through each task from beginning to end while working with SpecMan. Part II describes the step-by-step instructions that lead through each task from beginning to end while working with NMR-SAMS.

Q-2 (betulinic acid) (Fig. 1) is a natural triterpene isolated from American white-barked birches (Betula papyrifera Marsh., Betulaceae) and was provided by the group of Dr. N. Farnsworth in Univ. of Chicago. It exhibits a molecular formula C₃₀H₄₈O₃ (MW = 456) based on ¹³C-NMR and DEPT spectroscopy and EI-MS. Its identity with betulinic acid was proposed by comparing its physical data as well as NMR spectral data with those reported in the literature (S. Siddiqui et al., J. Nat. Prod. 51, 229 (1988); M. Sholichin et al., Chem. Pharm. Bull. 28, 1006 (1980).) Because of the severe overlap in resonances, there has been no report of complete ¹H and ¹³C assignments for this compound until the recent studies of the molecule at higher magnetic field (C. Peng et al, Magn. Reson. in Chem., 36, 267-278 (1998).).

Although this tutorial is organized in such a way that peak picking using SpecMan is described in Part I, and structure elucidation using NMR-SAMS is described in Part II, it is highly recommended that you run both programs side-by-side.

Figure 1. Two-dimensional structure of Q-2 with the ¹³C and ¹H (in parenthesis) resonance assignments. The numberings of the atoms corresponds to those of the ¹³C and ¹H peaks in Tables I and II.

We recommend users who do not use SpecMan, but work with other third party spectral analysis software, to manually edit the peak lists generated by their third party software to conform with NMR-SAMS format.

Part I: Computer-Assisted NMR Analysis and Assignment with SpecMan

This part provides step-by-step instructions for computer-assisted peak picking with SpecMan.

I-1. Transferring Processed Spectrum from NMR Spectrometers

The spectral data of Q-2 was acquired on a Varian Unity-plus 720 MHz spectrometer and processed (FT, phase correction, etc.) with VNMR 5.1. The concentration of the sample used was 0.084 M in pyridine-d₅. In order to import Varian processed spectra into SpecMan, one needs to transfer two files, namely, phasefile and procpar, for each spectrum to the working directory on the SGI workstation. For this example, first create Q-2 as main working directory, with the following sub-directories:

H-1/

C-13/

DEPT/

DQFCOSY/

HMQC/

HMBC/

NOESY/,

Next copy the phasefile and procpar files of each NMR experiment, into the corresponding sub-directories from the sample data directory described below.

The default location for the sample data of SpecMan and NMR-SAMS is: /usr/share/Spectrum/Data/SpecMan/Q-2 for SpecMan data, and /usr/share/Spectrum/Data/NMR-SAMS/Q-2 for NMR-SAMS data. The NMR experimental data is located in the sub-directories (H-1 through NOESY). Before proceeding with the rest of the tutorial, please make a backup copy of the original distribution data.

I-2. Analysis of 1D Proton Spectrum

At the UNIX prompt, type specman to launch the SpecMan program. Then, open the 1D file by selecting Open Spectrum option in the File menu. The following file browser dialog box appears: Select the File Type as Varian, then use the file browser to change directory to H-1 and double click on the phasefile in this directory.

After double clicking on the “phasefile” name in the right list box, the 1D spectrum will be displayed in a 1D Slice Window as shown below.

Setting Reference

The next step is to set the reference. Although the reference parameters are obtained from the procpar file, the user may still want to change the spectral reference. To zoom a peak, use the right mouse button to select the left top corner and drag the mouse (keeping the right mouse button pressed) to the desired right bottom corner (a rectangular zoom box will be drawn as you do this action) and release the button. The peak that you selected will be expanded and redrawn in the window. In this 1D spectrum you will set the reference on a small, weak peak due to CHCl₃at about 7PPM. To zoom this peak, keep the shift key on the keyboard pressed and then use the right while mouse button as described above to zoom this peak. You will notice the zoom being applied with fixed vertical scales. If the you want to further increase the vertical expansion of the peak keep the Ctrl key pressed while using the right mouse button for rubber-band zooming. This will help you adjust the vertical scales to the desired extent. For this spectrum, set the reference to the middle peak of Pyridine. After zooming this peak select the Set Reference option in the Edit Menu. Place the cursor at the top of the peak and click the left button. The following Set 1D Reference dialog box appears with the X chemical shift of the current location: Type 7.55 in the X Reference PPM.

Click OK to accept the new reference. To reset the expansion to full view, select Reset Zoom in the Display Menu (or press zoom icon in the tool bar). Once reference is set, the relevant spectral parameters will be saved with the procpar file.

In vast majority of cases peak picking of proton NMR spectrum is pointless, and without a very complicated analysis, one is not going to get what one needs, proton chemical shifts, from such a peak pick. For second order spectra the analysis becomes even more tedious. So it is not just a consequence of severe overlap in ¹H peaks which makes it difficult to do peak picking directly on this spectrum, but also other reasons as stated above. Hence, it is preferable to use 2D HMQC to extract 1D proton chemical shifts (see section I-5).

I-3 Analysis of 1D Carbon Spectrum.

Setting Reference and Appropriate Threshold

To begin analyzing the 1D carbon spectrum, open the “phasefile” file from the C-13 directory. Then, set reference in a similar manner as described for 1D ¹H. The reference is set on the middle peak of pyridine at 135.5ppm. Next, 1D peak picking of the ¹³C spectrum will be performed. Before peak picking, set the appropriate threshold. To do this, select the Set Threshold button in the 1D Control Panel (as seen below).

This will activate a red horizontal cursor on the 1D spectral window as the mouse is moved in this window. Move this horizontal bar to a suitable position so that it is just above the noise peaks. Then, click the left mouse button to update the threshold in the 1D Control Panel. One can also type the actual value in the threshold box. For this example use 9.906e-03 for threshold.

Automatic Peak Picking

Choose Pick Peaks Automatically from the Analysis menu, then choose 1D from the Pick Peaks Automatically pull-right menu. This activates the Pick 1D Peak dialog box as seen below.

Turn off the Negative Peak option and then click OK. After peak picking, 38 picked peaks are displayed on the spectrum, and are also listed in the 1D Peaks Table.

Manually Removing and Adding Peaks

Next, delete the three solvent peaks (triplets between 150 to 123 ppm; a total of nine peaks, numbered 28 through 36) by choosing the Remove Peaks from the Analysis menu. After activating Remove Peaks the program prompts the user to define a box around the peaks that need to be deleted. A rectangular rubber-band zoom box is defined to remove the solvent peaks. Do this by clicking the mouse in the upper right corner of the box to remove peaks from, and drag the mouse to the lower right corner. Release the mouse button to remove the peaks that are enclosed by the box. After removing the solvent peaks, deactivate the Remove Peaks button in the Analysis menu by selecting that option again.

If necessary, one can also add peaks by first selecting Add Peaks Manually from the Analysis menu and then choosing the Without Refine from the Add Peaks Manually pull-right menu. After selecting this option, click the cursor at the desired position to add a peak. For this example, no peak is added here. (Here we assume that at this stage the user has not identified the overlapping peak at 16.44 ppm. This will be pointed out by NMR-SAMS and the peak will be split into two peaks in the subsequent sections).

Sorting, Editing and Saving Peaks Table

Next select Edit Table button in the 1D peaks table. The following Edit Peaks Table dialog box will appear:

Complete the dialog box as follows: Select Sort Table Entries button. Select Descending radio button and X Value radio button to sort the table in the descending order of X Values (i.e. ¹³C Chemical shifts). Select the Renumber Table ID's button on. Then click OK to sort the table. The 29 peaks in the table are sorted and renumbered in the descending order of their chemical shifts.

Next click Save Table in the 1D peaks table, and type a filename c13 to save the peaks in a table called c13.pks. This table will be saved in the same sub-directory. (The extension ".pks" is automatically added).

I-4 Analysis of DEPT Spectra

The DEPT experiment usually consists of DEPT-45, DEPT-90, and DEPT-135. When you use Varian spectrometers, these are stored in the form of an arrayed 1D experiments and SpecMan automatically detects the multiple 1D spectra and offers a browser in the 1D Control Panel (as seen below) to allow the user to view each individual experiment one after another just like a normal 1D experiment. Also an option of Frozen Scale is provided to freeze both the ppm and intensity scales with respect to the previous spectrum. (This is a useful tool for comparing the build-up of peaks between different 1D spectra or slices). SpecMan also has an option to view multiple Spectra simultaneously. For instance you can open the ¹³C spectrum along with different DEPT spectra and display them in one window for comparison and analysis in multiple view mode. For more details regarding the usage of multiple view mode and options to tie these experiments for concurrent expansions, please refer to the SpecMan User’s Guide.

In order to get the ¹³C multiplicity information, only two of the experiments, DEPT-90 and DEPT-135, are necessary, although use of DEPT-45 may help to detect potential errors such as missing peaks. For this example, we do the peak picking for all three spectra, but use only the DEPT-90 and DEPT-135 data in the subsequent analysis with NMR-SAMS.

Setting Reference and Appropriate Threshold

The DEPT spectra are analyzed one at a time. After opening the phasefile from the DEPT directory, click and draw the slider in the 1D Control Panel to display the DEPT-45 spectrum for setting the reference (DEPT-45 is the first spectrum in the array, so the slider should be at the left-most edge, and should display ‘1’). The first peak from the left in the spectrum is selected to set the reference to 109.9134 PPM (same as the corresponding ¹³C peak) according to the following steps:

Select Peaks Table with pull-right 1D option in the Analysis menu. A 1D peaks table will be displayed (this could be an empty one if the previous peaks table was cleared and closed). Next click Load Table in the 1D Peaks Table panel and select c13.pks file in the C-13 sub-directory. The annotations of the ¹³C peaks (picked from the ¹³C 1D experiment ) will be overlaid on the DEPT spectrum. If the peak top of the first peak from the left in the DEPT-45 spectrum matches with the peak symbol( shown as “plus”) corresponding to ¹³C peak at 109.9134 PPM, then the reference is already set, and you can skip rest of this section to proceed with the peak picking step described in the next section. If the peak top is shifted from the ¹³C reference peak, then select Set Reference from the Edit Menu, and place the cursor on this peak. Next keeping the left mouse button pressed (a symbol in the form of a “plus” enclosed within a circle appears to mark the peak position selected for setting the reference), drag the cursor and release it at the center of the “plus” peak symbol which shows the position (which is the peak symbol at 109.9134 PPM). After releasing the left mouse button, a dialog box (similar to the one shown in Page 8) is displayed, showing the current chemical shift. Click OK and the reference of all the DEPT spectra will be the same as that of the ¹³C spectrum.

Peak Picking of DEPT-45 Spectrum

Next, perform peak picking of DEPT-45 spectrum. Set the appropriate threshold as described before (or type 1.098e-2 in the threshold box on 1D Control panel). Select Pick Peak Automatically with a pull-right of 1D from the Analysis menu and the program picks 22 peaks. Next sort the peaks in the peaks table in the descending order of X Value (i.e., ¹³C chemical shifts) as described previously. The sorted peaks are saved as dept45.pks by using the Save command in the 1D peaks table.

Peak Picking of DEPT-90 and DEPT-135 Spectra

Next process the DEPT-90 (which is the second spectrum array, so the slider in the ID Control panel is set at '2') and DEPT-135 (which is the fourth spectrum in the array, so the ID Control panel is set to '4') spectra in a similar way. For DEPT-90 use the threshold 2.577e-2 and 6 peaks will be picked by auto 1D peak picking. For DEPT-135 use the threshold 8.347e-3 and also turn the Negative Peaks button on in the 1D peak picking dialog box (because there are negative peaks in DEPT-135). 22 peaks will be picked in the DEPT-135 spectrum. As before Sort and Save the peaks as dept90.pks and dept135.pks, respectively.

I-5 Analysis of HMQC Spectrum.

HMQC spectrum provides both C-H direct connectivity information and the ¹H chemical shifts of carbon-attached protons. Although the latter information can be obtained from 1D ¹H spectrum if there aren’t many overlapping peaks, HMQC helps to resolve peak overlap and gives better separation for the ¹H peaks.

Setting Threshold

The phasefile of HMQC spectrum is opened by selecting Open Spectrum in the File menu. If the import is being performed for the first time, contours will be generated on the fly based on a computed threshold. The spectrum is displayed in the main SpecMan view window, along with the Threshold Control Palette, both as seen below.

The Threshold palette is used to set appropriate threshold, number of contour levels and the contour level separation. These controls can be adjusted interactively with the sliders. Before changing the threshold, turn off the Auto Redraw button in the Threshold palette. Auto redraw can be turned on when working with smaller data sets which are sub-matrices of a large 2D spectra. Another way to set appropriate threshold is by stepping through the Starting Level slider. Stepping through levels to determine the threshold is useful when the spectrum has severe t₁ or t₂ noise ridges.

After adjusting the starting level, click Update to re-generate the contours with the new threshold. Also one can adjust the Separation and Number of Levels to get a more satisfactory display of the peaks. For this spectrum, use Threshold as 3.548e-03, Separation as 1.3, and the Number of Levels as 20.

Setting Spectral Reference

Next, set spectral reference. On 2D spectra, SpecMan’s Associate Reference Spectra Option is useful for aligning 1D and 2D peaks. To use this feature, select the Associate Reference Spectra option in the Display menu. When this option is selected, a dialog box appears (as seen below) and prompts for the file names of reference spectra and reference peaks table. This option allows simultaneous display of 1D reference spectra on a 2D spectrum. The ¹H reference spectrum is displayed along the X axis (F2 dimension), and the ¹³C reference spectrum (or DEPT-45), as well as the ¹³C peak list, are displayed along the Y-axis (F1 dimension).

(The details of using this dialog box can be seen by selecting the Help button on this dialog box.) After selecting the appropriate reference spectra and the corresponding peaks table of ¹³C (you can click the Browse… buttons to graphically search for the appropriate files), click OK to display the reference spectra and the ¹³C grid lines (¹H peaks table is not available now; otherwise it can also be displayed as grid lines). The grid lines are drawn at the coordinates of the 1D ¹³C peaks and they will be used to verify the peak picking results later as seen below.

To set the reference of the HMQC, first zoom on a well-resolved cross peak with its corresponding 1D ¹H and ¹³C peaks displayed. Check if both the 1D ¹H and ¹³C peaks are aligned with the center of this cross peak. If the 1D’s are not properly aligned, you must set reference on 2D HMQC again to match these 1D peak locations. Select Set Reference in the Edit Menu and a cross hair cursor will appear. Move the cross hair cursor intersection to the center of the cross peak and press the left mouse button, and keeping it pressed (as soon as you press the left mouse button, the selected position is marked with a symbol that has a “plus” within a circle) drag it to the intersection point of the 1D ¹H and ¹³C peak coordinates in the 2D spectra so that it matches with the intersection, and release the left mouse button. The Set 2D Reference dialog box will appear (as shown below) with the new X and Y reference PPM for that location.

Click OK in the dialog box to accept these new reference values. This will set the reference on the selected cross peak.

Correcting Chemical Shift Reference Offset between 1D and 2D

It is important to verify the alignment of 1D and 2D peak coordinates. Do the following steps to verify and correct the alignment as needed. First zoom on the cross peak which is at the lower-most left corner of the 2D spectrum and check if both the ¹H and ¹³C peak are aligned well with the center of this cross peak. Set reference on that peak as described above and then zoom in on a cross peak which is at the upper-most right corner of the 2D spectrum. Check if both the ¹H and ¹³C peak are aligned well with the center of this cross peak. If there is a discrepancy, then the sweep width along X or Y needs to be adjusted. This correction in sweep width can be applied by selecting the Spectral Parameters option in the Edit Menu. Enter the new Sweep Width along X or Y in the dialog box that appears.

In this example the Sweep Width of both dimensions are adjusted (by trial and error) until the 1D and 2D peaks are well aligned. Click OK to change the spectral parameters.

Note that the modified parameters are saved by SpecMan so the next time the spectrum is opened, such correction does not need to be repeated.

Automatic Peak Picking

Peak picking of HMQC data consists of auto peak picking followed by a brief manual editing of the peak list. For this example, select Pick Peak Automatically in the Analysis menu with the 2D option in the Pick Peak Automatically pull-right menu. The following dialog box appears with various peak picking options:

Turn off the Negative Peaks button (SpecMan automatically turns this off for you if the Threshold Palette has the Negative contours turned off), and choose the Merge Peak Multiplets option to pick center of mass of cross peak multiplets. The Merge Peak Multiplets option can be performed in three modes: Average, Weighted Average and Highest Peak. Select the Weighted Average in the Multiplet Picking Option.

SpecMan peak picking algorithm uses peak width filters to discriminate noise from real peaks, and multiplets from independent peaks. A multiplet will be merged and the center of mass will be taken as the peak position. These filters are defined in terms of minimum and maximum box sizes for the search algorithm. A peak with width smaller than the minimum box size will be filtered as noise peaks, while the local extrema falling inside the maximum box size will be merged as the components of a multiplet. These limits can be set graphically by selecting the Set Graphically button in the peak picking dialog box. Upon selecting this option SpecMan will prompt the user to draw a rectangular box with the left mouse button on the peak which could be either a noise peak or a real cross peak for setting minimum and maximum limits respectively. For this data, enter 0.005 and 0.05 as Minimum X and Minimum Y for filtering noise peaks, and 0.05 and 1.0 as Maximum X and Maximum Y for merging multiplets. Next click OK to pick peaks. 33 peaks are automatically picked by SpecMan and the 2D Peaks Table will be displayed.

Manual Editing of Peak Picking Results

For efficient structure elucidation, it is always important to refine the peak picking results carefully. SpecMan provides a nice peak table interaction feature for the user to examine the zoomed 2D peaks one at a time. To do this the user first zooms on a particular peak of interest and then selects the peak entry in the 2D peaks table to browse through the next peak. The keyboard arrow keys can be used to step up and down in the table peak by peak. In this manner the user steps through every peak entry in the peaks table and SpecMan automatically zooms and displays the selected peak. While examining a zoomed peaks in the 2D spectrum, the user can compare its center with the grid lines which indicate the expected positions of the peak centers.

Here a few peak positions are corrected mostly by removing the automatically picked peak and adding one at the desired position. To remove a peak, activate Remove Peaks in the Analysis menu and then define a box around the peaks that need to be deleted. To add a peak, choose Add Peaks Manually from the Analysis menu with the pull-right option Without Refine, Singlets, or Multiplets, depending on what type of peak you are adding.

For this HMQC spectra, the Singlet option works well. This option allows you to click the left mouse button near the center of the peak, and SpecMan will automatically find the highest point. For more control over peak locations, you can use the Without Refine option, which adds the peak exactly where you click. The Multiplet Picking option picks the center of mass of the box region that you define. For this spectrum a few peaks were not picked at the real center of the multiplets because of their line shapes. You can select Move Peaks on the Analysis menu and move such peaks to the center of the multiplets. To move a peak, click on the peak label, and drag to the new location to release. Finally 33 peaks were obtained.

Sorting and Saving Peaks, Extracting ¹H Chemical Shifts

Click Edit Table button in the 2D Peaks Table and the following Edit Peaks Table dialog box appears:

In this dialog box, choose Sort Table Entries according to Descending, Y Value (i.e., ¹³C chemical shift) as the Sort Key and select Renumber Table ID's to renumber after the sort. Also, select Extract coordinates to 1D Table along the X dimension. After selecting the above options, click OK. SpecMan sorts the 33 HMQC peaks and creates a 1D peaks table of the 33 ¹H chemical shifts. Sort the ¹H peaks in the 1D peaks table by selecting Sort Table Entries according to Descending, X Value (i.e., ¹H chemical shift) as the Sort Key and select Renumber Table ID's to renumber peaks after the sorting. Next select the Save button in the 1D peaks table and use the file name h1 to save the 1D ¹H peaks. A file with h1.pks name will be saved in the current directory. Also, save the 2D HMQC peaks table as hmqc.pks.

Now the ¹H chemical shifts can be displayed as grid lines on the HMQC spectrum. To do this, select Associate Reference Spectrum in the Display menu, click the Browser following the 1D Reference Peak List along X, and choose h1.pks. Click OK, the ¹H grid lines will be displayed. Together with the ¹³C grid lines, the user can verify the peak picking results again. It is important to extract the ¹H chemical shifts as accurately as possible because they will be used for the peak picking of the other 2D spectra.

Adding Proton Peaks due to Heteroatoms

The ¹H peaks table extracted from the HMQC peaks table does not include the protons attached to heteroatoms. To make the ¹H peaks table more complete, open and display the ¹H spectrum as described previously, and load the ¹H peaks table by selecting Load Table button in the 1D peaks table. (Choose Peaks Tables with pull-right option 1D in the Analysis menu if the 1D Peaks Table is not already open ). The picked ¹H peaks are now labeled on the ¹H spectrum. The unlabelled peaks, which usually correspond to proton attached to heteroatoms, are added to the current ¹H peak list in the following manner. Choose Add Peaks Manually with the pull-right option Without Refine in the Analysis menu. A pointer cursor will appear (an icon of a hand with a pointer finger). Move the cursor with the mouse to the appropriate location where the unlabelled peak is and click the left mouse button to add a peak at that location. Repeat this step till you have added all the peaks. Next deactivate the add peaks mode by selecting again the Add Peaks Manually with the pull-right option Without Refine in the Analysis menu. Next save the modified peaks table with the same filename again.

For this example, the OH peaks are hardly visible and we don’t add any proton peaks from the heteroatoms.

I-6 Analysis of DQF-COSY Spectrum

Setting Spectral Reference and Threshold

Using procedures similar to the ones described for the analysis of HMQC, the DQF-COSY spectral file (phasefile) is opened from the DQF-COSY directory, displayed with appropriate threshold and spectral reference. The threshold for DQF-COSY is set at 7.546e-03, with Contour Separation as 1.4, and Number of Levels as 24. It is recommended to use a fairly low threshold of DQF-COSY so that all the weak COSY peaks appear. The reason for this is that NMR-SAMS uses the negative information from DQF-COSY to improve the efficiency of structure generation. For example, two proton-bearing carbons can be forbidden to connect if their protons show no DQF-COSY peaks. To take advantage of this feature, one has to make sure that all the COSY peaks, including the very weak ones, are picked.

Grid Intelligence-based Peak Picking

As described before in the HMQC analysis, select Associate Reference Spectrum from the Display menu to display the 1D ¹H reference spectrum along both X and Y axis. Also select the ¹H chemical shifts table (h1.pks) to display the grid lines on the DQF-COSY spectrum. Next, the spectral reference is calibrated in the same manner as the HMQC spectrum by checking the alignment of the 1D peaks with the 2D peaks. The Sweep Width parameters of both dimensions are adjusted to get a better alignment between the 1D and 2D peak coordinates.

The Peaks of DQF-COSY were picked with a novel peak picking method called “grid intelligence-based peak picking.” In this procedure, the grid intersection points, instead of the minimum peak width, are used as filter in the search algorithm for peak picking. After a multiplet has been merged and the center of mass is calculated using the current peak merge option, SpecMan attempts to locate a grid intersection point within the specified minimum peak width box size. If such a grid point is found, the peak center is retained as a real peak. Otherwise, it is rejected. To use this method for Q-2, perform the following steps:

Select Pick Peaks Automatically with 2D pull-right option in the Analysis menu. This will bring up a Pick 2D Peaks dialog box (as shown on page 21). Select both the Positive Peaks and Negative Peaks options. The Ignore Diagonal Peak option is not used as the diagonal peaks will be automatically removed by NMR-SAMS in the subsequent analysis. Next, check Grid Intelligence. Under Grid Distance Filter, set 0.008 for both Minimum X PPM and Y PPM. Alternately the Grid Distance filter can also be set graphically by using the Set Graphically button in the 2D peak picking dialog box. See SpecMan User’s Guide for details. Then select the Merge Peak Multiplets option and set the Multiplet Picking Option as Weighted Average to process multiplets. Under the Peak Width Filter, set 0.005 for both Minimum X PPM and Minimum Y PPM as limits for filtering noise peaks, and set 0.05 for both Maximum X PPM and Maximum Y PPM as the maximum peak width for considering a cross peak multiplet. Click OK, and 86 peaks will be picked in less than 1 minutes. For more details on using the Use Grid Intelligence method refer to the on-line help of SpecMan.

After the peak picking, it is important to examine the picked peaks and correct the ones which are either missed or not picked properly. The display of the reference 1D spectra as well as the grid lines facilitates the user’s verification of the peak picking results to a great extent. Note that in the subsequent analysis steps, NMR-SAMS will automatically discard the diagonal peaks and merge symmetry-related peaks for a homonuclear spectrum. So it is not necessary to discard the diagonal peaks at this stage. Although it is not very important to pick the symmetric peaks (on either side of the diagonal), picking both peaks may improve the reliability of the subsequent analysis. SpecMan picks both the symmetric peaks, and NMR-SAMS filters them appropriately during the conversion from SpecMan peak list to NMR-SAMS NMR data file. In cases where multiple grid centers are close to a peak and it is difficult to resolve as an unambiguous correlation, it is best advised to leave the picked peak as is, because NMR-SAMS will automatically include the different possible correlations to 1D peaks, and treat this cross peak as an ambiguous correlation information. In this spectrum, some noise peaks are removed from the peak list. Some of the ignored peaks are added (e.g., the peaks around (1.42, 1.20) and (1.37, 1.20)). Some incorrectly picked peaks which are significantly away from the grid centers are corrected. Finally, 87 peaks are obtained. The peaks table is sorted and renumbered with the Edit Peaks Table option, and then saved as a file (e.g. "cosy.pks"). If you have trouble cleaning the DQFCOSY data, you can load the cosy.pks peak list that was supplied with the sample data into SpecMan to see which peaks were retained. To do this, click Load Table… in the 2D Peak Table palette and select cosy.pks in the file browser. Then click OK and the clean COSY peaks will be annotated on the spectrum.

Note that, in addition to the parameters listed in the Pick 2D Peaks dialog box, the peak picking results also depend on the chemical shift reference and the alignment between the 1D and 2D spectra (when grid intelligence is used). If some of these are changed, you may get slightly different results, but it should not drastically affect the subsequent structure elucidation.

I-7. Analysis of HMBC Spectrum

Setting Spectral Reference and Threshold

Using procedures similar to the ones described for the analysis of HMQC, the HMBC spectrum is opened, displayed with appropriate threshold and spectral reference. The threshold for HMBC is set at 3.578e-03, with contour Separation as 1.2, and Number of Levels as 20.

As described before in the HMQC analysis, select Associate Reference Spectrum from the Display menu to display the 1D ¹H and ¹³C reference spectra along both X and Y axis, respectively. Also select the ¹H and ¹³C chemical shifts table (h1.pks and c13.pks) to display the grid lines on the HMBC spectrum. Then the spectral reference is calibrated in the same manner as the HMQC spectrum by checking the alignment of the 1D peaks with the 2D peaks. The spectral width of both dimensions are adjusted to get a better alignment between the 1D and 2D peak coordinates.

Grid Intelligence based Peak Picking

Similar to the DQF-COSY, the peaks of HMBC are picked with the grid intelligence-based method. To use this method do the following steps:

Select Pick Peaks Automatically with 2D pull-right option in the Analysis menu. This will bring up a Pick 2D Peaks dialog box (as shown on page 21). Turn off the Negative Peaks option and select the Grid Intelligence button. Under Grid Distance Filter set Minimum X PPM as 0.011, and Y PPM as 0.11. Next select the Merge Peak Multiplets option and set the mode as Weighted Average to process multiplets. Under Peak Width Filter, set 0.003 for Minimum X PPM, 0.05 for Minimum Y PPM as limits for discriminating noise peaks, and set 0.03 for Maximum X PPM and 0.5 for Maximum Y PPM. The maximum limits are used as the maximum peak width for considering a cross peak multiplet. Click OK, and 150 peaks will be picked in less than 1 minute. For more details on using the Grid Intelligence method refer to the on-line help of SpecMan.

I-8. Analysis of NOESY Spectrum

The display and peak picking of NOESY spectrum are done in a very similar manner as described above for the HMBC spectrum. Using procedures similar to the ones described for the analysis of HMBC spectrum is opened, displayed with appropriate threshold and spectral reference. The threshold for NOESY is set at 4.235e-03, with contour Separation as 1.2, and Number of Levels as 20.

As described before in the HMBC analysis, select Associate Reference Spectrum from the Display menu to display the 1D ¹H reference spectra along both X and Y axis, respectively. Also select the ¹H chemical shifts table (h1.pks) to display the grid lines on the NOESY spectrum. Then the spectral reference is calibrated in the same manner as the HMQC spectrum by checking the alignment of the 1D peaks with the 2D peaks. The spectral width of both dimensions are adjusted to get a better alignment between the 1D and 2D peak coordinates.

Similar to the DQF-COSY, the Peaks of NOESY are picked with the grid intelligence-based method. To use this method do the following steps:

Select Pick Peaks Automatically with 2D pull-right option in the Analysis menu. This will bring up a Pick 2D Peaks dialog box (as shown on page 21). Turn off the Negative Peaks option and select the Use Grid Intelligence button. Under Grid Distance Filter, set 0.008 for both Minimum X and Maximum X PPM. Next select the Merge Peak Multiplets option and set the mode as Weighted Average to process multiplets. Under Peak Width Filter, set 0.01 for Minimum X PPM, 0.01 for Minimum Y PPM as limits for discriminating noise peaks, and set 0.05 for Maximum X PPM and 0.05 for Maximum Y PPM. The maximum limits are used as the maximum peak width for considering a cross peak multiplet. Click OK, and 68 peaks will be picked in less than 1 minute. For more details on using the Use Grid Intelligence method refer to the on-line help of SpecMan.

NOESY is useful to NMR-SAMS only when the user opts to use the negative information of COSY together with NOESY. For example, if there is neither a COSY nor a NOESY peak observed between two proton-attached carbon atoms then this carbon pair is forbidden to connect. For Q-2 example we choose to use the negative information of COSY. The NOESY data is only used for creating and exporting the NOE assignments after the structure has been generated (for details see Section II-18). For this reason, the NOESY peaks table is not manually revised.

Part II Computer-Assisted Structure Elucidation by NMR-SAMS

II-1 Introduction

In this part, the SpecMan peak tables of ¹H, ¹³C, DEPT-45, DEPT-90, DEPT-135, 2D DQF-COSY, HMQC, HMBC, and NOESY spectra are first converted into correlation information between the 1D peaks. Such correlation information is then interpreted to define bond constraints on the atoms labeled by the 1D chemical shifts. These bond constraints are transformed into a set of mutually-consistent C-C bond constraints. Based on this information, a set of structural building blocks are generated, and an atom-atom connection matrix (ACMX) is setup. Finally the plausible 2D structures are generated. During the 2D structure generation, NMR-SAMS also completes the resonance assignments for candidate 2D structures which are complete and consistent with the NMR data. If complete 2D structures are not obtained during structure elucidation, then NMR-SAMS reports only the largest possible partial structure with resonance assignments. NMR-SAMS also provides resonance assignments for 2D structures that are proposed as possible target structures by the user.

II-2 Getting Started with NMR-SAMS

At the UNIX prompt, type nmrsams to launch the NMR-SAMS program. The program starts with a Main Graphics Window that has a menu bar and another window called Status Window which displays text prompts to indicate the current status of the structure elucidation. The messages in the status window also tells the user the next possible steps to do with NMR-SAMS. In most of the steps during the structure elucidation, NMR-SAMS displays messages which are of four types: error, warning, information and interrogation corresponding to different situations. For this part of the tutorial use the SpecMan generated peak lists provided under the sample data directory of NMR-SAMS which is located in: /usr/share/Spectrum/Data/NMR-SAMS/Q-2.

II-3 Opening New Working Data Set

By selecting New in the File menu, a file browser is brought up, listing all the files with extension of ".mdf" in the current sub-directory. Enter a new root name for the working data set as input (say Q-2-test).

After clicking OK, NMR-SAMS will create the following files in the current directory:

An empty master data file (MDF, with file extension .mdf), Q-2-test.mdf, where all the intermediate and final results are stored.

A default parameter file, called Q-2-test.par, where the control parameters used for the data interpretation and structure generation are stored. NMR-SAMS uses a set of default control parameters, but also provides the user a means to change these control parameters. This can be done by choosing the Parameters with pull-right options NMR Interpretation, Setting up ACMX and 2D Structure Generation in the Edit menu.

An empty NMR data file, called Q-2-test.nmr, where the NMR data converted from the SpecMan peaks table will be stored. The user can edit this file by choosing NMR Data File in the Edit menu.

An empty log file, called Q-2-test.log, where most of the information, warning, and error messages produced during the analysis will be stored. The user can view the log file by choosing Log File in the Edit menu.

An empty structure file, called Q-2-test.str, where the connection table of the generated structures and their resonance assignments will be stored. The user can display the structures by choosing the Generated Structures option in the Display menu.

A lock file, Q-2-test.lock, which is used to prevent two users opening the same data set simultaneously.

Next the user is prompted to enter the molecular formula via the Molecular Formula dialog box (shown below). For this example, enter C30H48O3.

After clicking OK, the molecular formula is automatically interpreted for element composition and common valences. These are written into the MDF after the keyword “ATOMS:”.

Note: If a certain atom has unusual valence, specify the valence after the element symbol. Otherwise the common valence will be adopted for it. Examples are “C30H47N(V)O32”, and “C30H48N(V)NO32”, where N(V) indicates a nitrogen atom with valence of 5.

Note: If you do not know the molecular formula, you can type “unknown”. For structure elucidation with unknown molecular formula, please see section II-18.

II-4 Conversion of SpecMan ¹H Peak List.

First select Create NMR Data File with the pull-right option H-1 in the File menu, NMR-SAMS will open the following dialog box with prompt to enter the filename of the ¹H peaks table from SpecMan (In this case, select the h1.pks file).

After entering the file name Click OK and an information dialog box (shown on next page) displays that 33 ¹H peaks have been read.

Click OK again and another information dialog box appears (shown below) prompting the user to supply the ¹H multiplicity for the peaks.

Click OK to accept this message and then select the option NMR Data File in the Edit menu to open Q-2-test.nmr in vi editor mode. (If the user prefers jot editor, he can choose jot in the initial file nmrsams.ini and then restart the program.) Edit this file as shown in table I by replacing the unknown multiplicities “u” of 8 singlet peaks (#s 1, 2, 12, 25, 28, 29, 30 and 32) with "s". After editing save the changes by exiting the vi editor in the usual manner. The remaining ones, which are difficult to discern, are left as “unknown”(u). Table I shows these changes.

Note that NMR-SAMS recognizes only the multiplet patterns such as singlet (s), doublet(d), triplet(t), and quartet(q). All others must be entered either as unknown (u) or multiplet (m) in general. The multiplet information will be used to eliminate inappropriate bonds during structure generation (Refer to section II-14) for the rules.

Table I. The ¹H peak list of Q-2

H1: /usr/people/peng/NMR-SAMS/ndat/Q-2-test/h1p.pks

#1. 4.930 s ;1

#2. 4.755 s ;2

#3. 3.509 u ;3

#4. 3.435 u ;4

#5. 2.725 u ;5

#6. 2.611 u ;6

#7. 2.235 u ;7

#8. 2.232 u ;8

#9. 1.924 u ;9

#10. 1.863 u ;10

#11. 1.830 u ;11

#12. 1.778 s ;12

#13. 1.752 u ;13

#14. 1.653 u ;14

#15. 1.565 u ;15

#16. 1.544 u ;16

#17. 1.540 u ;17

#18. 1.513 u ;18

#19. 1.439 u ;19

#20. 1.419 u ;20

#21. 1.370 u ;21

#22. 1.369 u ;22

#23. 1.368 u ;23

#24. 1.249 u ;24

#25. 1.208 s ;25

#26. 1.196 u ;26

#27. 1.196 u ;27

#28. 1.062 s ;28

#29. 1.051 s ;29

#30. 0.993 s ;30

#31. 0.969 u ;31

#32. 0.818 s ;32

#33. 0.811 u ;33

The first line which begins with the keyword “H1:” indicates the start of ¹H peak list. After the keyword “H1:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Peak ID, chemical shift, multiplicity, and comments (optional) for each ¹H peak, respectively. One or more space(s) is used as a delimiter for all items except comments which are separated by “;” Comments can always be included as long as they follow a “;”. In this table, the number in the comment field corresponds to the ID of peak in the SpecMan peaks table. The comments are not used by NMR-SAMS.

II-5 Conversion of SpecMan ¹³C and DEPT Peak List.

Upon selecting Create NMR Data File with pull right option ¹³C and DEPT in the File menu, the following dialog box is displayed. Click Browse after “SpecMan C-13 Peaks Table” and select c13.pks in the file browser. Select DEPT as “Peak Multiplicity Experiments”. Turn off DEPT-45 Peaks Table since DEPT-45 experiment provides redundant information as the other two experiments do. Click Browse… after “DEPT-90 Peaks Table” to select file dept90.pks. Click Browse… after “DEPT-135 Peaks Table” to select file dept135.pks. Also the user is prompted to input a matching tolerance between DEPT and ¹³C peaks, as shown in the following dialog box.

After entering the file name and 0.08 PPM for the tolerance, Click OK and an information dialog box (as shown below) displays that 29 ¹³C peaks have been read (see Table II).

Click OK again and the peak list is checked with the molecular formula. By comparison with the molecular formula, the program warns the user that there are fewer ¹³C peaks than expected (as the molecular formula has 30 carbons), as shown in the following dialog box.

Click OK to accept this warning message and then select the option NMR Data File in the Edit menu, to open file Q-2-test.nmr in vi editor mode. (If the user prefers jot editor, he can choose jot in the initial file nmrsams.ini and then restart the program.) From the ¹³C peak height, as well as the number of corresponding cross peaks in the HMQC spectrum, it is easy to identify ¹³C peak #27 (16.441ppm, q) as a degenerate one. So another peak (#30) with slightly different chemical shift (16.442 ppm) is added at the end of the ¹³C peak list. To do this, copy the line of peak #27 and paste it at the end of the peak list. Then renumber it as peak #30 and modify the chemical shift from 16.441 to 16.442. This peak is also marked as a split peak (The comment and peak intensity are not used by NMR-SAMS). After editing save the changes by exiting the vi editor in the usual manner. The remaining ones, are left unchanged. Table II shows these changes.

(Note: In the case of a symmetric molecule or severe ¹³C overlapping, one can only input the well-resolved portion of the ¹³C peaks and NMR-SAMS will perform partial structure elucidation based on the available spectral data. Only substructures will be generated then.)

Table II. The ¹³C peak list of Q-2

C13: /usr/people/peng/NMR-SAMS/ndat/Q-2-test/c13.pks

#1. 178.822 s ;1

#2. 151.323 s ;2

#3. 109.931 t ;3

#4. 78.147 d ;4

#5. 56.647 s ;5

#6. 55.956 d ;6

#7. 50.997 d ;7

#8. 49.814 d ;8

#9. 47.783 d ;9

#10. 42.877 s ;10

#11. 41.151 s ;11

#12. 39.540 s ;12

#13. 39.318 t ;13

#14. 38.648 d ;14

#15. 37.596 t ;15

#16. 37.556 s ;16

#17. 34.868 t ;17

#18. 32.901 t ;18

#19. 31.246 t ;19

#20. 30.301 t ;20

#21. 28.679 q ;21

#22. 28.330 t ;22

#23. 26.149 t ;23

#24. 21.243 t ;24

#25. 19.507 q ;25

#26. 18.813 t ;26

#27. 16.441 q ;27

#28. 16.340 q ;28

#29. 14.929 q ;29

#30. 16.441 q ;27 Split from #27

The first line which begins with the keyword “C13:” indicates the start of ¹³Cpeak list. After the keyword “ C13:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Peak ID, chemical shift, multiplicity, and comments (optional) for each ¹³C peak, respectively. One or more space(s) is used as a delimiter for all items except comments which are separated by “;”. In this table, the number in the comment field corresponds to the ID of peak in the SpecMan peaks table. The comments are not currently used by NMR-SAMS.

II-6 Conversion of SpecMan COSY Peak List.

Upon selecting Create NMR Data File with pull right option COSY in the File menu, the user is prompted to enter the filename of the COSY peaks table from SpecMan (cosy.pks). Also the user is prompted to input a matching tolerance between 1D and 2D coordinates for each dimension, as shown in the following dialog box:

After entering the file name and 0.005 PPM for the tolerances (along X & Y), Click OK and a warning message dialog box (see below) is displayed to warn the user about ambiguous correlation for some peaks.

Click OK To All button, and the program displays an information dialog box (shown below) indicating that 32 COSY correlations were obtained. Then the user is warned to denote some of the potential long-range coupled peaks as "weak" ones, because the program by default assumes all COSY as short-range (geminal and vicinal) coupling. The user can also supply accurate J-coupling constants, and let the program determine nature of coupling (long-range or short-range) based on that information.

For this example, click OK and select the NMR Data File option in the Edit Menu to edit the NMR data file (.nmr file) in vi mode. (If the user prefers jot editor, he can choose jot in the initial file nmrsams.ini and then restart the program.) Modify the intensity level of 6 peaks (#s 1, 2, 3, 8, 20 and 29) in the COSY peaks from “3” (i.e., strong, the default value) to “1” (i.e., weak), and save the changes with the save and exit command. (See Table III). These 6 peaks are identified mainly from there weak intensities as due to potential long-range coupling.

(Note: A potential long-range coupling COSY peak is usually interpreted as 3-5 intervening bonds between the correlated protons, which also covers the possibility of vicinal coupling. A short-range coupling is usually interpreted as 2-3 intervening bonds between the correlated protons. If a long-range coupling is mistakenly interpreted as a short-range one, NMR-SAMS will not generate the correct structure. So it is very important to denote these potential long-range coupling peaks. The geminal coupling is always automatically detected by the program.)

Table III. The COSY peak list of Q-2

COSY:

#1. (1 - 2) 1 0.00 ;1+4

#2. (1 - 12) 1 0.00 ;2+31

#3. (2 - 12) 1 0.00 ;3+32

#4. (3 - 7 8) 3 0.00 ;6+18

#5. (3 - 13) 3 0.00 ;7+33

#6. (3 - 18) 3 0.00 ;5+49

#7. (4 - 11) 3 0.00 ;8+30

#8. (5 - 9) 1 0.00 ;12+21

#9. (5 - 13) 3 0.00 ;11+34

#10. (5 - 26 27) 3 0.00 ;82+87

#11. (6 - 10) 3 0.00 ;15+26

#12. (6 - 16 17) 3 0.00 ;14+83

#13. (6 - 24) 3 0.00 ;13

#14. (7 8 - 15) 3 0.00 ;17+38

#15. (7 8 - 18) 3 0.00 ;16+48

#16. (9 - 20) 3 0.00 ;22

#17. (9 - 26 27) 3 0.00 ;20

#18. (10 - 16 17) 3 0.00 ;27+47

#19. (10 - 24) 3 0.00 ;24

#20. (10 - 28) 1 0.00 ;23

#21. (11 - 14) 3 0.00 ;29+36

#22. (11 - 31) 3 0.00 ;28

#23. (14 - 31) 3 0.00 ;37+75

#24. (16 17 - 19) 3 0.00 ;51+85

#25. (16 17 - 21 22 23) 3 0.00 ;43+55

#26. (16 17 - 24) 3 0.00 ;45+60

#27. (16 17 - 33) 3 0.00 ;84

#28. (19 - 21 22 23) 3 0.00 ;52+58

#29. (19 - 29) 1 0.00 ;50

#30. (20 - 26 27) 3 0.00 ;53+64

#31. (21 22 23 - 26 27) 3 0.00 ;56+71

#32. (21 22 23 - 33) 3 0.00 ;86

The first line which begins with the keyword “COSY:” indicates the start of COSYpeak list. After the keyword “COSY:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Cross Peak ID, IDs of the correlated 1D ¹H peaks shown in parenthesis (for ambiguous correlations the IDs of all possible 1D ¹H peak correlations are included), peak intensity levels (which are classified as four types; strong, medium, weak, and unknown, and denoted as 3,2,1 and 0 respectively. The default value is always 3.), J-coupling (optional, the default value is 0), and comments (optional, with a maximum size of 80 characters), for each COSY cross peak, respectively. For a short range coupled DQF-COSY peaks intensity levels should be either 3 or 2. For long-range coupled DQFCOSY peaks, the intensity levels should be 1. If an intensity level 0 used, the program will expect actual J-coupling values in the field which represents J-coupling. One or more space(s) is used as a delimiter for all items except comments which are separated by “;”. Items marked as optional can be omitted unless an item following it is included. In such a case, please include default values for ignored items even if they don’t get used. Comments can always be included as long as they follow a “;”. In this table, the numbers in the comment field correspond to the IDs of the peaks in the SpecMan peaks table. For merged peaks these numbers are shown with a + sign. The comments are not currently used by NMR-SAMS.

II-7 Conversion of SpecMan HMQC Peak List.

Upon selecting Create NMR Data File with the pull-right option HMQC (or HETCOR) in the File menu, the user is prompted to enter the filename of the HMQC peaks table from SpecMan (hmqc.pks). Also the user is prompted to input a matching tolerance between 1D and 2D coordinates for each dimension as shown in the following dialog box below. After entering the file name, and 0.005 PPM as tolerance for matching ¹H peaks (along X) and 0.1 PPM as tolerance for matching ¹³C peaks (along Y), Click OK.

A warning message dialog box (shown below) is displayed to warn the user about a ¹³C peak with fewer correlations than expected. Click OK button to ignore the message, and 33 HMQC peaks are obtained and stored in the NMR data file. (see Table IV).

(Note: Unlike other 2D peaks, where ambiguous correlations are allowed, the HMQC peaks must have exactly two correlated 1D peaks).

Table IV. The HMQC peak list of Q-2

HMQC:

#1. (3 - 1) ;2

#2. (3 - 2) ;1

#3. (4 - 4) ;3

#4. (6 - 33) ;4

#5. (7 - 21) ;5

#6. (8 - 13) ;6

#7. (9 - 3) ;7

#8. (13 - 14) ;8

#9. (13 - 31) ;9

#10. (14 - 5) ;10

#11. (15 - 7) ;12

#12. (15 - 15) ;11

#13. (17 - 19) ;14

#14. (17 - 23) ;13

#15. (18 - 6) ;16

#16. (18 - 17) ;15

#17. (19 - 8) ;18

#18. (19 - 18) ;17

#19. (20 - 10) ;19

#20. (20 - 24) ;20

#21. (21 - 25) ;21

#22. (22 - 11) ;22

#23. (23 - 9) ;24

#24. (23 - 26) ;23

#25. (24 - 20) ;25

#26. (24 - 27) ;26

#27. (25 - 12) ;27

#28. (26 - 16) ;28

#29. (26 - 22) ;29

#30. (27 - 32) ;31

#31. (28 - 30) ;32

#32. (29 - 28) ;33

#33. (30 - 29) ;30

The first line which begins with the keyword “HMQC:” indicates the start of HMQCpeak list. After the keyword “HMQC:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Cross Peak ID, IDs of the correlated 1D ¹³C & ¹H peaks shown in parenthesis, and comments (optional, with a maximum size of 80 characters), for each HMQC cross peak, respectively. One or more space(s) is used as a delimiter for all items except comments which are separated by “;”. The comments are not currently used by NMR-SAMS.

II-8 Conversion of SpecMan HMBC Peak List.

Upon selecting Create NMR Data File/HMBC (or COLOC) in the File menu, the user is prompted to enter the filename of the HMBC peaks table from SpecMan (hmbc.pks). Also the user is prompted to input a matching tolerance between 1D and 2D coordinates for each dimension (as shown below). Here 0.005 PPM and 0.08 PPM are used as tolerance for ¹H and ¹³C peaks respectively.

During the conversion, some warning messages appear indicating (as shown in the dialog box below) that ambiguous cross peaks are obtained. Click OK to All to ignore similar messages for this example.

Other warning messages appear (as shown in the dialog box below) when a SpecMan peak is discarded because its X (¹H) or Y (¹³C) chemical shift does not match any 1D peak. This happens if it is an artifact or the center of a peak is not accurately located. Click Help for suggestions. For this example, click OK to All to ignore the similar messages.

After the conversion, 129 HMBC peaks (Table V) are obtained and saved in the NMR data file after a keyword “HMBC”. It can be seen that some peaks have ambiguous correlation to 1D peaks due to very close chemical shifts.

Note: Although NMR-SAMS can use ambiguous correlation information, too many ambiguous correlations will undermine the efficiency of the subsequent structure generation. So whenever possible the user should resolve ambiguities manually.

SpecMan provides a nice peak table interaction feature to examine the zoomed 2D peaks one by one. To do this the user first zooms on a particular peak of interest and then selects the peak entry in the 2D peaks table to browse through the next peak. In this manner the user steps through every peak entry in the peaks table and SpecMan automatically zooms and displays the selected peak. While examining the zoomed peak, in the 2D HMBC spectrum, the user must compare its center with the intersection of grid lines which indicate the positions of the 1D ¹H and ¹³C chemical shifts. If the ¹H/¹³C chemical shifts are too close to resolve, the user must retain these peaks as ambiguous correlation. In this example, there are 36 ambiguous cross peaks which are not well resolved.

Table V. The HMBC peak list of Q-2

HMBC:

#1. (1 - 6) ;3

#2. (1 - 7 8) ;4

#3. (1 - 13) ;5

#4. (1 - 15) ;2

#5. (1 - 16 17) ;1

#6. (2 - 1) ;9

#7. (2 - 2) ;11

#8. (2 - 3) ;8

#9. (2 - 8) ;6

#10. (2 - 12) ;7

#11. (2 - 18) ;10

#12. (3 - 3) ;12

#13. (3 - 12) ;13

#14. (4 - 11) ;14

#15. (4 - 14) ;16

#16. (4 - 25) ;17

#17. (4 - 30) ;15

#18. (5 - 5) ;25

#19. (5 - 6) ;20

#20. (5 - 7 8) ;26

#21. (5 - 10) ;24

#22. (5 - 13) ;21

#23. (5 - 15) ;22

#24. (5 - 16 17) ;23

#25. (5 - 18) ;19

#26. (5 - 24) ;18

#27. (6 - 14) ;30

#28. (6 - 19) ;32

#29. (6 - 21 22 23) ;27

#30. (6 - 25) ;31

#31. (6 - 30) ;29

#32. (6 - 32) ;28

#33. (7 - 9) ;33

#34. (7 - 20) ;38

#35. (7 - 21 22 23) ;37

#36. (7 - 29) ;35

#37. (7 - 31) ;36

#38. (7 - 32) ;34

#39. (8 - 3) ;43

#40. (8 - 5) ;41

#41. (8 - 6) ;42

#42. (8 - 7 8) ;40

#43. (8 - 16 17) ;39

#44. (9 - 1) ;45

#45. (9 - 2) ;44

#46. (9 - 7 8) ;46

#47. (9 - 12) ;49

#48. (9 - 13) ;48

#49. (9 - 15) ;47

#50. (9 - 18) ;50

#51. (10 - 5) ;57

#52. (10 - 6) ;54

#53. (10 - 9) ;55

#54. (10 - 10) ;56

#55. (10 - 13) ;59

#56. (10 - 19) ;51

#57. (10 - 24) ;52

#58. (10 - 28) ;53

#59. (10 - 29) ;58

#60. (11 - 10) ;65

#61. (11 - 16 17) ;63

#62. (11 - 19) ;60

#63. (11 - 20) ;64

#64. (11 - 21 22 23) ;61

#65. (11 - 28) ;66

#66. (11 - 29) ;62

#67. (12 - 4) ;69

#68. (12 - 11) ;67

#69. (12 - 16 17) ;72

#70. (12 - 25) ;70

#71. (12 - 30) ;68

#72. (12 - 33) ;71

#73. (14 - 9) ;79

#74. (14 - 13) ;78

#75. (14 - 20) ;76

#76. (14 - 24) ;75

#77. (14 - 26 27) ;80

#78. (14 - 28) ;77

#79. (15 16 - 7 8) ;81

#80. (15 16 - 11) ;89

#81. (15 16 - 13) ;90

#82. (15 16 - 14) ;87

#83. (15 16 - 16 17) ;88

#84. (15 16 - 18) ;84

#85. (15 16 - 21 22 23) ;85

#86. (15 16 - 31) ;83

#87. (15 16 - 32) ;82

#88. (15 16 - 33) ;86

#89. (17 - 16 17) ;92

#90. (17 - 21 22 23) ;91

#91. (17 - 29) ;93

#92. (17 - 33) ;94

#93. (18 - 10) ;97

#94. (18 - 13) ;98

#95. (18 - 15) ;96

#96. (18 - 24) ;95

#97. (19 - 3) ;99

#98. (19 - 7 8) ;101

#99. (19 - 15) ;100

#100. (20 - 6) ;104

#101. (20 - 16 17) ;102

#102. (20 - 28) ;103

#103. (21 - 4) ;106

#104. (21 - 30) ;105

#105. (21 - 33) ;107

#106. (22 - 14) ;109

#107. (22 - 31) ;108

#108. (23 - 5) ;111

#109. (23 - 13) ;112

#110. (23 - 26 27) ;110

#111. (24 - 5) ;116

#112. (24 - 21 22 23) ;115

#113. (24 - 26 27) ;114

#114. (25 - 1) ;120

#115. (25 - 2) ;119

#116. (25 - 3) ;118

#117. (26 - 19) ;122

#118. (26 - 21 22 23) ;123

#119. (26 - 32 33) ;121

#120. (27 28 30 - 4) ;130

#121. (27 28 30 - 25) ;129

#122. (27 28 30 - 32 33) ;128

#123. (27 30 - 14) ;126

#124. (27 30 - 19) ;124

#125. (27 30 - 21 22 23) ;127

#126. (27 30 - 31) ;125

#127. (29 - 5) ;132

#128. (29 - 10) ;133

#129. (29 - 24) ;131

The first line which begins with the keyword “HMBC:” indicates the start of HMBCpeak list. After the keyword “HMBC:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Cross Peak ID, IDs of the correlated 1D ¹³C & ¹H peaks shown in parenthesis (for ambiguous correlations the IDs of all possible 1D ¹³C & ¹H peaks are included ), and comments (optional, with a maximum size of 80 characters), for each HMBC cross peak, respectively. One or more space(s) is used as a delimiter for all items except comments which are separated by “;”. The comments are not currently used by NMR-SAMS.

II-9 Conversion of SpecMan NOESY Peak List

Upon selecting Create NMR Data File/NOESY (or ROESY) in the File menu, the user is prompted to enter the filename of the NOESY peaks table from SpecMan (noesy.pks). Also the user is prompted to input a matching tolerance between 1D and 2D coordinates for each dimension. Here 0.005 PPM are used as matching tolerance for both dimensions respectively. This procedure is very similar to that described in II-8. (Click OK to All to all the warning messages) After the conversion, 49 NOESY peaks are obtained and saved in the NMR data file after a keyword “NOESY”. The peaks are shown in Table VI.

Table VI. The NOESY peak list of Q-2

NOESY:

#1. (1 - 2) 3 0.16 ;1+2

#2. (1 - 3) 3 0.01 ;4

#3. (1 - 12) 3 0.01 ;19

#4. (2 - 12) 3 0.01 ;20

#5. (3 - 7 8) 3 0.01 ;13

#6. (4 - 25) 3 0.01 ;60

#7. (4 - 32) 3 0.01 ;88

#8. (5 - 29) 3 0.02 ;75

#9. (6 - 16) 3 0.02 ;9

#10. (6 - 17) 3 0.01 ;33

#11. (7 8 - 15) 3 0.05 ;11+27

#12. (7 8 - 18) 3 0.04 ;35

#13. (9 - 20) 3 0.01 ;43

#14. (9 - 26 27) 3 0.03 ;14

#15. (10 - 24) 3 0.07 ;15+56

#16. (10 - 29) 3 0.02 ;74

#17. (11 - 14) 3 0.01 ;25

#18. (11 - 32) 3 0.02 ;87

#19. (12 - 25) 3 0.01 ;62

#20. (13 - 16 17) 3 0.01 ;32

#21. (13 - 28) 3 0.01 ;21

#22. (14 - 20) 3 0.01 ;24+42

#23. (14 - 31) 3 0.01 ;23

#24. (14 - 32) 3 0.01 ;89

#25. (15 - 21 22 23) 3 0.01 ;26

#26. (16 - 21 22 23) 3 -0.02 ;51

#27. (16 17 - 25) 3 0.01 ;59

#28. (16 17 - 28) 3 0.01 ;71

#29. (16 17 - 32 33) 3 0.01 ;91

#30. (19 - 21 22 23) 3 0.04 ;52

#31. (19 - 28) 3 0.02 ;69

#32. (19 - 32) 3 0.01 ;84

#33. (20 - 26 27) 3 0.07 ;40+66

#34. (20 - 32) 3 0.01 ;85

#35. (21 - 28) 3 0.02 ;45

#36. (21 22 - 30) 3 0.01 ;78

#37. (21 22 23 - 24) 3 0.01 ;50

#38. (21 22 23 - 30) 3 0.02 ;48

#39. (21 22 23 - 31) 3 0.01 ;47

#40. (21 22 23 - 32 33) 3 0.02 ;46

#41. (22 23 - 33) 3 0.02 ;93

#42. (24 - 28) 3 0.01 ;70

#43. (25 - 30) 3 0.03 ;77

#44. (25 - 32) 3 0.03 ;86

#45. (25 - 32 33) 3 0.01 ;63

#46. (26 27 - 28) 3 0.02 ;68

#47. (29 - 32) 3 0.04 ;83

#48. (30 - 32) 3 0.09 ;79+90

#49. (31 - 33) 3 0.01 ;94

The first line which begins with the keyword “NOESY:” indicates the start of NOESYpeak list. After the keyword “NOESY:”, following a blank space comments may be added up to 80 characters in length. The entries in the rest of the lines represent the Cross Peak ID, IDs of the correlated 1D ¹H peaks shown in parenthesis (for ambiguous correlations the IDs of all possible 1D ¹H peak correlations are included), peak intensity levels (which are classified as four types; strong, medium, weak, and unknown, and denoted as 3,2,1 and 0 respectively. The default value is always 3.), actual peak intensity or volume (optional, the default value is the peak intensity), and comments (optional, with a maximum size of 80 characters), for each NOESY cross peak, respectively. One or more space(s) is used as a delimiter for all items except comments which are separated by “;”. Items marked as optional can be omitted unless an item following it is included. In such a case, please include default values for ignored items even if they don’t get used. Comments can always be included as long as they follow a “;”. In this table, the numbers in the comment field correspond to the IDs of the peaks in the SpecMan peaks table. For merged peaks these numbers are shown with a + sign. For NOESY peak list the peak intensity levels are used for calibrating NOE distance bounds in the NOE assignments.

II-10 Generation of Building Blocks

When you select Building Blocks from the Analysis menu, NMR-SAMS interprets the molecular formula, ¹³C, ¹H, and HMQC spectral data, and generate possible building blocks for structure generation. All data except ¹³C spectral data are optional. This procedure consists of the following three successive steps.

Interpretation of MF:

The interpretation of molecular formula is automatically done when the molecular formula is first entered after opening a new working data set. But the molecular formula is interpreted again once you select Building Blocks from the Analysis menu. The molecular formula is interpreted for element composition. These results are written into the MDF after the keyword “ATOMS:”.

Note that for each step, NMR-SAMS keeps only one copy of the results in the master data file (.mdf file ). If any step is repeated, the previous results of that step, as well as the results generated by the dependent steps subsequent to it, are overwritten. If a dialog box prompts about this, Click Yes to overwrite.

Interpretation of ¹H, ¹³C, and HMQC Spectral Data

After interpreting the molecular formula, NMR-SAMS reads the ¹H, ¹³C, and HMQC peak lists in the NMR data file (.nmr file) and saves the results of interpretation in the master data file (.mdf file). In the following sections the results of NMR data interpretation are described for each type of peak list.

¹H Peaks: The chemical shifts and the multiplicities are listed in the MDF after a keyword "1DH1". The integral information is not used now.

¹³C Peaks: The chemical shifts and number of attached protons (derived from the ¹³C multiplicity) are listed in the MDF after a keyword "1DC13". The number of ¹³C peaks is compared with that of the constituent carbon atoms to determine the symmetry of the molecule, if the molecular formula is known.

Note that in the case of a symmetric molecule or severe overlap of ¹³C peaks, where fewer ¹³C peaks than the constituent carbon atoms are observed, NMR-SAMS will only consider the carbon atoms labeled by the observed ¹³C peaks, and generate partial structures (or substructures). This is called "partial structure elucidation".

HMQC Peaks: Each peak is interpreted as one-bond connectivity between the relevant ¹³C and ¹H spins. Here 33 bond constraints are listed in the MDF after a keyword "HMQC".

If a ¹H peak is found to have no HMQC peak, the user will be prompted to supply the type of heteroatom attached to it. The program then automatically assigns a heteroatom to the proton and adds a bond constraint together with the HMQC ones. If the user chooses not to add such connectivity between the ¹H peaks and heteroatoms, all other connectivity information (e.g. COSY and HMBC) concerning these ¹H peaks will be ignored and this could result in increased computation time, and more plausible candidate structures. For Q-2 example, since no OH peaks were picked, the message regarding heteroatoms does not appear, and therefore this does not apply.

Generating Building Blocks

This step performs the task of allocating protons to the heavy atoms according to the ¹³C multiplicities. Each of the resulting structural fragment, such as -CH₃ and -OH, will be used as structural building blocks in the subsequent structure generation. The unsatisfied valences of each building block, determined from its chemical valence and number of attached protons, are referred to as free bonds. In the subsequent structure generation, the program will connect the free bonds and get the structure.

For Q-2 one building-block set is obtained and stored in the MDF after the keyword "FRAG_SET". Note that sometimes multiple building-block sets result from the permutation of protons on the heteroatoms. In such cases, it is recommended to remove the undesired ones. (Click Delete on the browser to delete the currently displayed one, or click Select to delete all but the current one). NMR-SAMS can deal with multiple building-block sets for structure generation, but currently uses only the first one for resonance assignment.

The building blocks are displayed graphically. By default, the ¹³C and ¹H chemical shifts assigned to each building block are displayed graphically as well as in a connection table. You can select Display Options in the Display menu to change the displayed attributes.

II-11 User-Defined Building Blocks

NMR-SAMS provides a function that allows you to edit the building blocks generated by the program as described above. This function is especially useful when the molecular formula is unknown. In that case, NMR-SAMS will automatically deduced the carbon building blocks (e.g. -CH3, >CH2) from the ¹³C peaks and multiplicities. Then you can add heteroatoms as building blocks.

To edit building blocks, choose User-Defined Building Blocks from the Analysis menu. The follow palette is displayed:

To add a building block, select Add, type the symbol of the heavy atom, select the number of attached protons, define its valence, and click on the main graphic window. A building block will be added there.

To modify a building block, select Modify. Then click on the building block (which you want to modify) while pressing the Ctrl key. The attribute of that atom will be updated in the palette. Next change the attributes you want in the palette, and click on that building block again. The attributes will be updated for that building block.

Tip: If you just want to define some building blocks as “ignored” ones (see the tutorial for Paclitaxel), you can check Ignored Atom in the palette, and directly click on those building blocks one after another. The program will define them as ignored building blocks without changing the other attributes. If you click twice on a building block, then the program will update the other attributes.

To delete a building block, select Delete, then click on the building block you want to delete.

Note: User-defined Buiding Blocks function is mainly designed for you to edit the heteroatom building blocks. There are some restrictions on carbon building blocks. For example, you can not delete a carbon building block that was derived from a ¹³C peak.

II-12 Setting up Bond Constraints

After choosing Bond Constraints in the Analysis menu, NMR-SAMS interprets all available 2D spectral data (except HMQC) as bond constraints, and setup ACMX(s) for structure generation. The control parameters for spectral interpretation can be adjusted by by selecting the Parameters with pull-right option NMR Interpretation from the Edit menu. The control parameters for setting up ACMX can be adjusted by selecting the Parameters with the pull-right option Setting up ACMX in the Edit menu. The default values are used here.

This procedure consists of the following four successive steps.

Interpretation of 2D Spectral Data

NMR-SAMS uses the available peak list in the NMR data file (.nmr file) and interprets them as bond constraints between the correlated atoms. The results of interpretation are saved in the master data file (.mdf file). In the following sections the results of NMR data interpretation are described for each type of peak list.

COSY Peaks: The bond constraints regarding the correlated ¹H spins are listed in the MDF after a keyword "COSY" according to the interpretation controls specified by the user in the dialog box regarding NMR interpretation. This can be accessed by selecting the Parameters with pull-right option NMR Interpretation from the Edit menu. For this example, 6 potential long-range coupled COSY peaks are interpreted as 3-5 intervening bonds between the correlated protons. The remaining short-range coupled ones are interpreted as 2-3 bonds.

During the interpretation of COSY peaks, NMR-SAMS also warns the user to avoid some common pitfalls that may lead to incorrect structure generation. If two ¹H peaks are very close and no COSY peak is observed between them, the user is alerted to check if any near-diagonal peak has been neglected between them. If the user is not sure about this, the program allows the user to add a "pseudo bond constraint" for this proton pair. (The tolerance for checking near-diagonal COSY peaks can be changed by the user by selecting Parameters with pull-right option NMR Interpretation from the Edit menu. The default value is 0.02ppm). The following dialog box appears during interpretation of COSY:

For the COSY data of Q-2, Click Yes to All to add pseudo bond constraints for the following seven ¹H pairs: 7-8, 16-17, 19-20, 21-22, 21-23, 22-23, and 26-27.

HMBC Peaks: Each peak is interpreted as a bond constraint of 2 or 3 intervening bonds on the relevant ¹³C and ¹H spins. Here 129 bond constraints are listed in the MDF after a keyword "HMBC".

NOESY Peaks: Each NOESY peaks is interpreted as a bond constraint of 2 to 6 intervening bonds on the relevant ¹H spins. NMR-SAMS uses NOESY in a very limited fashion as described in the previous sections. NOESY bond constraints are not used for 2D structure generation.

Transforming Various BCs into C-C BCs.

In this step the various bond constraints, e.g. the H-H BCs from COSY and the C-H ones from HMBC, are transformed into C-C bond constraints based on the HMQC-derived C-H connectivity. NMR-SAMS also cross-checks the bond constraints for mutual consistency, and alerts the user whenever a contradiction is encountered. The bond constraints are written into the .mdf file after the keyword “C13~~C13”.

The format of a bond constraint (BC) is described below. A BC is represented in one line with the following information:

(Atom_y ... - Atom_x ... : minBond ~ maxBond; BondType; minNSBC ~ maxNSBC)Source

where

Atom_y ... is the correlated atom(s) along the Y dimension (¹³C domain for a heteronuclear spectrum). It could be more than one in the case of ambiguity.

Atom_x ... is the correlated atom(s) along the X dimension (¹H domain for a heteronuclear spectrum). It could be more than one in the case of ambiguity.

minBond and maxBond are the minimum and maximum bond separations between the relevant atoms.

BondType is the type of the intervening bond between the atoms: 0, 1, 2, or 3 for unknown, single, double, and triple, respectively.

minNSBC and maxNSBC are the minimum and maximum numbers of relevant atom pair(s) that must be satisfied for this BC.

Source encodes the cross peaks (or other source) from which the BC was derived. A cross peak is represented by its spectral type and its ID number. The following codes are used to represent the spectral types:

“C” for COSY, “Q” for HMQC (or HETCOR), “B” for HMBC (or COLOC), “N” for NOESY, “I” for INADEQUATE.

The following codes are used to represent the other kind of sources:

“S” for a pseudo BC added by the program, “U” for a user-define BC, and “G” for a previously generated bond (when using a previously generated substructure as the starting point for next structure generation).

During this process, NMR-SAMS save warning messages in the .log file when different bond separation or other properties are assigned to the same relevant atoms. It also warns, if the relevant atoms of a bond constraint are found to be the sub-set of another bond constraint. Normally this would not cause any problems during the 2D structure generation, but the user is advised to check any potential errors in the 2D peak lists. You can choose Log File from the Edit menu to open the .log file and check such warning messages.

When this task is completed a unified set of 157 C-C bond constraints are obtained and listed in the MDF after the keyword “C13~~C13”. One may note that some of the BCs arise from 10 or more individual cross peaks. For example, the following C-C bond constraint:

(18 - 20: 1 ~ 1; 1; 1 ~ 1)C15+26Q16Q19C13Q16Q20B97Q19B95Q20B104Q16

means that there must be a bond between C18 and C20, and this is derived from the 13 cross peaks denoted following the right parenthesis, where “C15+26” denotes the symmetric COSY peaks #15 and #26, “Q16” HMQC peaks #16 and so on. (The numberings of the peaks correspond to the peak IDs in the SpecMan peaks tables.).

During the bond constraint transformation, COSY correlations between geminal protons are automatically discarded. Moreover, to avoid overlooking a correct structure, if a ¹H peak is found to correlate with multiple ¹³C peaks, this ¹H peaks is taken as a degenerate peak and a pseudo Bond Constraint is added between the corresponding carbons. The is due to the fact that correlation between degenerate proton are generally not observed.

Setting up Atom-Atom Connection Matrix (ACMX)

This step performs the task of defining the bonding possibilities between the building blocks based on the available bond constraints. The unambiguous bond constraints (one bond between exactly two atoms) are treated as fixed bonds, and the rest are used as constraints during the subsequent structure generation. Generally, before selecting Bond Constraints in the Analysis menu to begin this process, it is important to ensure that the right set of parameters are used for setting up an ACMX. To modify the parameters, select the menu item Parameters with pull-right option Setting up ACMX from the Edit menu. A dialog box appears as shown below. The following list of parameters (rules) need to be taken care of:

1. Use of the COSY Negative Information: If the first button, Treat as Ideal Spectrum, is selected, then NMR-SAMS treats COSY as an ideal spectrum, i.e., two proton-bearing carbon atoms are forbidden to connect if no COSY peak is observed between them. Although this is usually true, and this reduces the time taken to generate structures, it could also lead to losing a correct structure if some ³J_H,H couplings were not observed for reasons such as H-H configuration or chemical environments.

If the second button, Use with NOESY Data, is selected (the default setting), then NMR-SAMS will use the negative information in conjugation with NOESY data. In this case, two proton-bearing carbon atoms are forbidden to connect if neither COSY nor NOESY peaks were observed between them. This is preferred over the previous choice and is recommended if NOESY data is available.

If the last button, Do Not Use, is selected, then the negative information is not used. In this case, all proton-bearing atoms will be allowed to connect even if no COSY peaks were observed between them. Though this is a safe option, this could significantly reduce the efficiency of subsequent structure generation.

2. Extract Unambiguous 1-Bond Constraints as Fixed Bonds: This flag defines the usage of NMR-derived unambiguous bond constraints (e.g. those from well-resolved COSY peaks) as fixed bonds prior to structure generation. Once a fixed bond is defined, it cannot be broken though its bond type can be changed in the subsequent structure generation. While this enhances the efficiency of structure generation, the correct structure may be lost if one of the fixed bonds is incorrect. (e.g., a long-ranged DQF-COSY peak was mistakenly interpreted as a vicinal coupling). The default is to use 1-bond constraints as fixed bonds.

If one chooses not to use the 1-bond constraints as fixed bonds, all NMR-derived bond constraints will be used during structure generation. In that case the bond constraints can be violated. But this may significantly reduce the efficiency of structure generation. Note that NMR-SAMS always treats user-supplied unambiguous bond constraints as fixed bonds. The user can set an upper limit to the number of violated bond constraints in this dialog box.

3. Use of ¹H Multiplicities to Suppress Inappropriate Bonds: If this option is selected (this is the default setting), the following rules are used to exclude some carbon atoms from bonding during the structure generation:

1. Only CHx-CHy (x > 0, y ³ 0) are considered;

2. CH₃ with a multiplicity M = 1(s), 2(d), 3(t), or 4(q) is forbidden to bond to CHy if y ¹ M -1;

3. CH₃ with other multiplicity’s M > 4 is forbidden to bond to CHy if y = 0;

4. CH with a multiplicity M = 1 is forbidden to bond to CHy if y = 2 or 3.

If this option is not selected, the structure generation may take longer to complete.

For this example, the Default values are used for all the parameters. Click on Default to set parameters and accept the dialog box by clicking OK.

Then select Set up ACMX in the Analysis menu and one ACMX will be set up. A summary of this process is displayed in a dialog box as shown below. Click OK to accept it.

The building blocks, as well as some fixed bonds are displayed as seen below. An atom with unsatisfied valence is marked by a “*” and displayed in different color (blue by default). The fixed bonds with unknown bond types are displayed as dashed lines. A fixed bond with an unknown bond type can become either a single, double, or triple bond after the subsequent structure generation. For this example, the carboxylic group is correct. For this example, the types of all fixed bonds are certain based on the ¹³C chemical shifts of the relevant carbon atoms.

As described in the Section II-13, the user can choose Display Options in the Display menu to change the displayed features. For example, you can select Display Options with pull-right option Show Disconnectivities from the Display menu. Then by clicking an atom in the displayed structure, the other atoms that can not be connected to the selected atom will be highlighted (the default color is cyanic). Also you can interact the display atoms with the bond constraints listed in the Connection Table. By clicking a bond constraint in the Connection Table, the relevant atoms are highlighted in the displayed structure (the default color is pink). By click an atom in the displayed structure, the relevant entries in the Connection Table are highlighted.

Note that NMR-SAMS automatically sets up a carboxylic acid group based on the ¹³C chemical shift of C-1 and the available heteroatoms. Sometimes the automatically added functional groups may not be correct. In such a case you can modify them by selecting User-Defined Bond Constraints from the Analysis menu.

II-13 User-Defined Bond Constraints

Before submitting the bond constraints and ACMX to NMR-SAMS for structure generation, it is important to verify the building blocks, fixed bonds, and the available bond constraints. For complex molecules, if the user has apriori knowledge about the structure, then additional constraints can be defined in the form of “user-defined bond constraints”. User-defined bond constraints concerning heteroatoms (O, N etc.) derived from other spectral data are especially important for improving the efficiency of the subsequent structure generation (shorter computational time and fewer candidate structures). Normally connectivity information concerning these atoms is not available from 2D NMR spectra, but the user can use additional information such as functional groups from IR, UV or MS data to define the non-NMR constraints.

NMR-SAMS allows the user to define bond constraints based on a set of building blocks, or a (sub)structure if structure generation has already been done. For this example, as the structure generation has not been done yet, display the building block set by selecting Building Blocks and Fixed Bonds in the Display menu.

Next select User-Defined Bond Constraints in the Analysis menu. A User-Defined Bond Constraints editor palette (shown below) is displayed. With this editor the user can add or delete bonds between the building blocks. For this example you do not need to add any more bond constraints because the program already automatically added the carboxylic group. Click Cancel to dismiss the palette.

In the current version of NMR-SAMS one cannot graphically use the User-Defined Bond Constraints editor to define ambiguous bond constraints in the form of either more than one single bond separation or ambiguous correlated atoms (e.g., correlation either between atom 1 and 2, or 1 and 3). However, the user can manually include these kind of constraints by editing the MDF file to explicitly add these ambiguous bond constraints. The user defined bond constraints are always specified after the keyword "ATOM~~ATOM" in the MDF file. Next select User-Defined Bond Constraints from the Analysis menu, and click OK in the User-Defined Bond Constraints palette. The manually added bond constraints in the MDF file will be used to re-setup the ACMX.

II-14 User-Defined Atom Environment Constraints

In addition to user-defined bond constraints, NMR-SAMS also allows the user to define constraints on the neighboring patterns of an atom, which is referred to as “atom environment constraints”. For example, if one knows that C-4 (based on its chemical shift 78.147) must have one oxygen as its neighbor, but does not know to which oxygen atom it should connect, then it can be defined as an environment constraint of C-4 as follows:

Select Atom Environment Constraints in the Analysis menu. An Edit Atom Environment Constraints palette (shown below) appears.

Type “4”, the ID of C-4, as Focus Atom ID. Type “O” (the default value) as the Neighboring Element. Select Single as the Bond Type between C-4 and the oxygen atom. Type “1” for both Minimum and Maximum Occurrences. This defines only one single-bonded oxygen atom to be the neighboring atom of C-4. Then click Add at the bottom of the palette to add this environment constraint. For Q-2, only the environment constraint described above is used. Then click OK to set up the ACMX again with this additional environment constraint included.

In a similar manner the user can add, modify, or delete other environment constraints.

As described in section II-15, all the bond constraints and fixed bonds will be cross-checked for mutual consistency before setting up the ACMX again. Click OK to All to ignore all the messages and a summary of the ACMX will be displayed again, indicating that the user-defined atom environment constraint is included. Click OK to accept the message. The building blocks and the fixed bonds are then displayed, and the connection table is updated with the environment constraint. (The ACMX itself is not displayed but can be inspected by choosing Master Data File in the Edit menu).

II-15 2D Structure Generation

One of the major bottlenecks in computer-assisted structure elucidation is the efficiency of structure generation (which is factor of the computation time, the quality of structure generated, and the number of candidate structures generated). The structure generator searches all the plausible 2D structures that are consistent with the data. If the spectral data is precise and has fewer ambiguities, then NMR-SAMS usually generates the correct unique structure within reasonable computation time. However, as structure elucidation is combinatorial problem, it becomes more and more time consuming as the size of the molecule and the number of free bonds increases (for example molecules with sizes > 30 heavy atoms). Several heuristic rules are applied to speed up the process of structure elucidation. Some of these controls can be adjusted by the user by selecting the option Parameters with pull-right option Structure Generation in the Edit menu. A dialog box will be displayed and the details of the parameters in the dialog box can be seen by selecting the on-line help. For this example the default values of all the parameters are used.

Next select Generate 2D Structures in the Analysis Menu to start the structure generation process. A Structure Generation in Progress dialog box is displayed (as shown below). This dialog box shows the initial state (in terms of molecular formula, number of free bonds and number of bond constraints used) and the current state (in terms of the completed structures) of the structure generation. The dialog box is updated at a frequency based on the parameter Interval for Updating Structure Generation Dialog Box. The default value is 0.1 minute, and you can change this parameter by selecting Parameters with pull-right option Parameters for 2D Structure Generation in the Edit menu).

At any time the user can abort the structure generation process by clicking the Stop button. It takes a few seconds to take effect after the Stop button has been clicked. The complete structure generation of Q-2 takes less than 1 minute of CPU time and the correct unique structure is obtained. After the completion of structure generation a dialog box (shown below) prompts the user to store the substructure along with the complete one. Click Yes to save the structures.

Then a dialog box (shown below) displays a brief summary of the results of structure generation. Click OK.

The structure is displayed along with a structure browser (shown below) which enables the user to browse through the remaining structures/substructures one at a time. Together 49 largest substructures are generated during this process are also recorded in the structure file (.str file). The substructures are sorted in the descending order of the number of generated bonds. The substructures provide additional clues in case where a complete unique structure has not been generated.

The candidate structures can be displayed with the carbon atoms labeled by their assigned ¹³C and ¹H chemical shifts. By selecting Display Options in the Display menu, the user can also choose to display a Connection Table of the structures. The status of each NMR-derived or user-supplied bond constraint, whether satisfied or violated, is also summarized in the table.

II-16 Exporting NMR Data, 2D Structures, and Resonance Assignments

NMR-SAMS provides an export function for NMR peak lists (in the form of chemical shift correlations). To create chemical shift correlation table, Select Export with pull-right option Chemical Shift Correlations in the File menu. The correlation of chemical shifts are written into a file (Q-2-test.spc) in a format that is familiar to the chemists.

NMR-SAMS provides a tool to export 2D structures to third party molecular drawing programs such as Chemdraw, ChemSketch etc. The structure is exported with coordinates in MDL format. To export structures, select Export option with the pull-right option Structure (MDL) in the File menu, and the currently displayed structure will be exported into a file “Q-2-test00x.mdl”, where x is the sequential number of the (sub)structure.

To export the result of resonance assignment, select the option Export with pull-right option Assignment in the file menu. The resonance assignments of the candidate structure or substructure which is on display will be written to a text file “Q-2-test00x.rst”, where x is the sequential number of the (sub)structure. This file contains the ¹³C and ¹H assignments of all the atoms in the molecule. If NOESY peaks are available, then the assignment of the NOESY peaks along with distance constraints and the actual bond separation between the relevant protons are included. This information enables resolving of ambiguous NOE peaks, and identification of through-space NOE connectivities.

II-17 Target Structure-based Resonance Assignment

Input of User-Proposed Target Structure

If the 2D structure elucidation has been successfully completed by the previous steps, then it is not necessary to perform this task. For instance we don’t need this for Q-2 because the generated structure has a complete resonance assignments. This option is only used when the user has some apriori knowledge about the structure of molecule in study, and is interested in knowing the assignments. Unlike other methods of assignment which are only based on predicting ¹³C or ¹H chemical shifts from large spectral databases, NMR-SAMS uses mainly 2D NMR-derived connectivity information for resonance assignment. During the assignment process, NMR-SAMS first uses some standard chemical shifts to predict tentative assignments. Next the 2D NMR-derived connectivity information is included to improve these tentative assignments to final resonance assignments. In this manner, the final assignments of NMR-SAMS are usually more reliable than the assignments based solely on predicted chemical shifts from large databases.

Suppose the user knows the structure of Q-2 and is only interested in its resonance assignments, then the following steps are executed:

After setting up the bond constraints (section II-14), build the proposed molecule by selecting Input Target Structure with pull-right option Build Molecule in the Analysis menu. This will bring a molecular editor palette (shown below) for interactive sketching of the molecule. If the proposed structure has already been built with a third party software, then first save it in MDL format, and then select Input Target Structure with Import MDL pull-right option in the Analysis menu to import the structure. This can be further modified with the Molecular Builder shown below.

After inputting the target structure, Click OK to accept this target structure. If previous results of structure generation exist in the MDF, the user is prompted to remove the results. Click Yes to remove previous results.

For a target structure manually sketched by the user, NMR-SAMS prompts the user (as shown below) to export it into an MDL file in case it’s entry in the MDF has been overwritten by some previous operations. Click OK to accept this message. Then NMR-SAMS stores the connection table of the target structure and the predicted ¹³C chemical shift ranges in the MDF. These results are located after the keyword "TSS".

To export the target structure into an MDL file, Choose Target Structure/Assignments in the Display menu to display the target structure. Next choose Export with pull-right Structure (MDL) option in the File menu. The target structure is written into an MDL file, Q-2-test000.mdl.

Next CISOC-SES automatically sets up an assignment matrix. This matrix summaries a preliminary assignment of the building blocks to the heavy atoms in the target structure based on the elemental types and observed and predicted ¹³C chemical shifts. ¹H chemical shifts are not used while building this matrix.

Resonance Assignment

Select Assign Spectra in the Analysis menu to begin the resonance assignment process. In this process, the building blocks are mapped to the constituent heavy atoms in the target structure using all of the available constraints. For Q-2, one complete assignment is obtained in a few seconds, and the following dialog box appears and prompts the user to save the partial assignments along with the complete assignment. Click Yes to save the partial assignments.

These assignments are exported as a text file in a format which is usually used by chemists.

Finally the following dialog box gives a brief summary of the results of resonance assignment. Click OK.

For Q-2, one complete assignment and 49 largest partial assignments are obtained. The option of saving the largest partial assignments is useful when complete assignment is not possible. After the assignments are completed, the structures can be displayed with the assignments. For multiple candidate assignments an Assignment Browser (as shown below) is used to step through the different possible assignments. The resonance assignment, structures, as well as the NMR data, can be exported into text files as described in Section II-17.

During resonance assignment NMR-SAMS tries to assign resonances to the best possible extent even if complete assignment is not possible. The resonance assignment starts from a selected atom (internally the program selects this based on a number of criteria), and if complete assignments is successfully obtained during the first attempt, NMR-SAMS stops after searching all the possible mappings. On the other hand, if a complete assignment is not obtained during the first attempt, NMR-SAMS loops through different starting atoms and repeats the assignment process in order to get the largest possible partial assignments. Such partial assignments can be displayed as substructures after the resonance assignment process is completed. By inspecting the unassigned portions of the substructures, the user may be able to identify the regions in the target structure which conflict with the NMR data. As it may take a long time to loop through all the starting atoms, the user is advised to use the Stop button to abort the process after a few starting atoms have been tried.

Analog Structure-Based Resonance Assignment

Usually the chemical shifts of analog structures are very similar. If one of them has been assigned, its assignment can be used as the starting point for the assignment of the other chemical cousins. This is called Analog Structure-based Resonance Assignment.

To do this, you have to export the target structure of the first molecule along with its assignments into a .mdl file. First select Target Structure/Assignments in the Display menu to display the assignment. Then select the assignment you want by using the Structure Browser. Next select Export with pull-right option Structure (MDL) from the File menu. The structure and the displayed assignment will be exported to the .mdl file.

For the next molecule, you can import first the target structure and its assignments by choosing Input Target Structure with the pull-right option MDL File from the Analysis menu. The previous assignments will be displayed, and NMR-SAMS will prompt you with the following dialog box:

If you want to use the previous assignment as the starting point, leave the toggle Analog-Based Assignment on. Then modify the C-13 Matching Tolerance if you like. Using the matching tolerance (default value: 3.0 ppm), NMR-SAMS compares the carbon chemical shifts of the assigned analog molecule with the corresponding ¹³C chemical shift of the current molecule to complete the first level of assignments for carbon atoms. Note that only the ¹³C chemical shift and multiplicity are considered. ¹H chemical shifts and 2D connectivities are not considered. So this function must be used with caution. During the automatic assignment process, the initial assignments will be retained.

If you want to use only the target structure, but not the previous assignments, de-select the Analog-Based Assignment toggle. Then click OK.

If you do not want to use the target structure at all, click Cancel.

User-Defined Resonance Assignment

NMR-SAMS allows you to edit the resonances assigned before or after performing automated resonance assignment. To do that, select User-Defined Resonance Assignment from the Analysis menu. A User-Defined Resonance Assignment palette is displayed as follows:

In the palette, the current resonance assignment is displayed. To add an assignment, select Add in the palette, then select the ¹³C ID in the list (which has a “None” as Assigned Atom #), and click a carbon atom in the displayed structure. After comparing the real and predicted ¹³C chemical shifts, as well as the peak multiplicity, NMR-SAMS will either accept or reject the assignment.

To delete an assignment, select Delete in the palette. Then click the ¹³C ID in the list, or click the atom which is associated with this ¹³C ID in the displayed structure. In either cases the assignment of this chemical shift will be removed from the atom.

By clicking Undo in the palette you can undo the last action.

Upon completing adding/deleting assignments, click OK to accept the changes, or Cancel to discard the changes. The current assignments can then be exported or used as the starting point for next automated assignment. To do that, choose Assign Spectra from the Analysis menu.

II-18 Structure Elucidation With Unknown Molecular Formula

If you do not know the molecular formula, you can still do the structure elucidation using NMR-SAMS. The working data set Q-2-nomf is an example. The procedure is mostly the same as in the case where molecular formula is known. The main differences are described as follows:

1. While creating the data set, input molecular formula as ‘unknown’ (see II-3).

2. During the peaks table conversions (see II-4-II-9), the program will not check the results against the molecular formula. So some of the warning messages will not appear.

3. During generating building blocks, it will generate building blocks according to the observed ¹³C peaks. So for this example, the heteroatoms will not be generated compared with those obtained in section II-10:

4. During User-Defined Building Blocks (see II-11), you have to add the heteroatom. As shown below, select Add in the palette, type “O” as Element, turn off Ignored Atom, make sure Proton Count is 0 and Valence is 2. Next click in the main graphics window one time to add an oxygen atom.

Next, as shown below, select 1 as the Proton Count, and click in the main graphics window 2 times to add 2 OH groups.

Up clicking OK in the palette, NMR-SAMS rearranges the building blocks and automatically sets up the ACMX again. The resulting building blocks should be the same as those described in II-10.

For the rest of the steps (following Section II-10), the operations and results should be exactly the same as those described for the example with a known molecular formula.

Computer-Assisted Structure Elucidation of Q-2 Using SpecMan and NMR-SAMS

Computer-Assisted Structure Elucidation of Q-2 Using SpecMan and NMR-SAMS

Computer-Assisted Structure Elucidation of Q-2
Using SpecMan and NMR-SAMS

Computer-Assisted Structure Elucidation of Q-2
Using SpecMan and NMR-SAMS