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Acknowledgments

Computer-Assisted Structure Elucidation of Paclitaxel (Taxol)
Using SpecMan and NMR-SAMS

Overview:

The following document provides step-by-step instructions that lead the user through the process of Computer-assisted Structure Elucidation of the Paclitaxel (Taxol, Fig.1) molecule. We assume the users of this document to have a good understanding of general NMR techniques and their application to structure elucidation problems. There are two parts to this document: Part I describes the step-by-step instructions for each task in SpecMan, while Part II describes the step-by-step instructions for each task in NMR-SAMS.

Figure 1. Two-dimensional structure of Paclitaxel (taxol) 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. The three phenyl groups are not considered during the computer-assisted structure elucidation.

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 the user run both programs side-by-side.

Part I: 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 Taxol sample was provided by Dr. R. Holton in the Chemistry Dept. of Florida State University. All of the 1D and 2D NMR data for this molecule was collected on a Bruker DMX 300 MHz spectrometer. Solvent CDCl₃ was used in all experiments. Some of the spectra were obtained from different sample conditions, and therefore slight chemical shift differences are observed between the spectra. In order to import Bruker processed spectra into SpecMan, the user needs to transfer the 1r/2rr files along with their corresponding procs and proc2s files to the working directory on the SGI/SUN workstation.

For this tutorial, the sample data is located in the Data directory on the CD. Copy the Data.tar file from the Data directory into the …/Spectrum2001 directory on your SGI/SUN. Next, untar the tar file ('tar xvf Data.tar') and the Data files will be untarred into the current directory. The default location for sample data is: …/Spectrum2001/Data/SpecMan/Paclitaxel for SpecMan data and …/Spectrum2001/Data/NMR-SAMS/ Paclitaxel for NMR-SAMS data. The following subdirectories will appear under the …/Spectrum2001/Data/SpecMan/Paclitaxel folder:

H-1

C-13

DEPT-45

DEPT-90

DEPT-135

HMQC

DQFCOSY

HMBC

I-2. Analysis of 1D Proton Spectrum

Make sure to be in the …/Spectrum2001/SpecMan directory, and type 'specman24' at the UNIX prompt to run SpecMan. Then, open the 1D ¹H file by selecting ‘Open Spectrum’ from the File menu. Select the File Type as Bruker, and then use the file browser to change to the H-1 directory and double click on the 1r file in this directory as shown below:

After double clicking on ‘1r,’ the 1D spectrum will be displayed in a 1D Slice Window as shown below:

Setting Reference

The next step in analyzing the ¹H spectrum is to set the reference. The original reference parameters are obtained from the procs file, but the user may want to change the spectral reference. In this case, zoom on the small, weak peak due to CHCl₃at about 7.27 ppm by using 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 selected peak will be expanded and redrawn in the window. Next, select ‘Set Reference’ from the Edit menu. Place the cursor at the top of the peak and click the left button. The following ‘Set Reference’ dialog box will appear with the X chemical shift of the peak:

If the value is not already 7.27, then type ‘7.27’ in the X Reference ppm text box, and click, ‘OK’.

To reset the expansion to full view, select Reset Zoom in the Display Menu (or click on the ‘Reset Zoom’ icon on the tool bar). Once the reference has been set, the relevant spectral parameters (including reference) will be saved in the procs file.

In a vast majority of cases, peak picking of ¹H NMR spectra is futile, due to severe overlap in the ¹H peaks. Therefore, it is preferable to use 2D HMQC to extract the 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 ¹³C spectrum, select open ‘1r from the C-13 directory and the following ¹³C spectrum will appear:

Similar to how the reference was set for the ¹H spectrum, set the reference on the CHC1₃ peak at 77.22 ppm. Next, set an appropriate threshold by selecting the ‘Set Threshold’ button from the 1D Control Panel, as shown below:

This will display a red horizontal line on the ¹³C spectral window. Move this line to a position just above the noise peaks and then left-mouse click to update the threshold value. One can also type the necessary threshold value in the threshold textbox on the 1D Control Panel (use 2.381e+07 for this example).

Automatic Peak Picking

To perform peak picking, select, ‘Pick Peaks Automatically’ from the Analysis menu with the pull-right option of ‘1D.’ The following dialog box will appear:

Uncheck ‘Negative Peaks’ and then click ‘OK.’ Forty peaks will be picked and displayed on the spectrum, and listed in the 1D Peaks Table as shown below:

Manually Removing and Adding Peaks

Next, delete the three solvent peaks (18, 19 and 20) by selecting, ‘Remove Peaks’ from the Analysis menu. Using the left mouse button, define a rectangular rubber-band box around the solvent peaks. As soon as the left mouse button is released, the solvent peak labels will no longer be displayed on the spectrum, and the peak entries will also be removed from the 1D Peaks Table. Next, re-click on the ‘Remove Peaks’ button from the Analysis menu to deactivate the option.

Next, zoom in on the large peak at 167.25 ppm, and notice that it is an envelope of two overlapping peaks. SpecMan enables the user to add a peak here by selecting ‘Add Peaks Manually’ from the Analysis menu with the pull-right option of ‘Without Refine.’ Then, click the left mouse button at the place where the smaller peak can be seen affecting the shape of the dominant peak, and a new peak will be added at this location. Finally, select ‘Add Peaks Manually – Without Refine’ again to deactivate the option.

Sorting, Editing and Saving Peaks Tables

Next select ‘Edit Table’ from the 1D peaks table, and the following ‘Edit Peaks Table’ dialog box will appear:

Select the following options:

‘Sort Table Entries’

‘Descending’

‘X Value (i.e. sort the table in the descending order of ¹³C Chemical shifts)’

‘Renumber ID’s’

‘Remove Redundant Peaks’

Then, click ‘OK’ to sort the table, and the 38 peaks will be sorted and renumbered in the descending order of their chemical shifts. Next click ‘Save Table’ from the 1D Peaks Table, and save the peaks table as ‘c13’ (The extension *.pks will be added automatically).

I-4. Analysis of DEPT Spectrum

The DEPT experiment usually consists of DEPT-45, DEPT-90, and DEPT-135. In order to obtain ¹³C multiplicity information, it is usually only necessary to analyze two of the three DEPT experiments: DEPT-90 and DEPT-135. Since DEPT-45 may sometimes be used to detect potential errors such as missing peaks, we will perform peak picking for all three spectra.

SpecMan allows the user to view all three experiments at one time by selecting, ‘Open Multiple Spectra’ from the File menu. This will bring up the following ‘open multiple spectra’ dialog box:

To add the first DEPT experiment, click on ‘Add’ and the following dialog box will appear:

Next, select the 1r file from the DEPT-45 folder. To add additional spectra, re-select ‘Add’, and then select the 1r file from the DEPT-90 folder and the 1r file from the DEPT-135 folder. As you select the files in the ‘Open’ file browser, the list of selected files will be displayed in the ‘Open Multiple Spectra’ dialog box shown below:

Once you have selected the desired spectra, click ‘OK’ in the Open Multiple Spectra dialog box and the following multiple spectra will be displayed:

When comparing DEPT spectra it is convenient to tie the axes display. This is accomplished through the ‘Tie Views’ option in the Display menu. When this option is chosen, the following ‘Tie Manager’ dialog box is displayed:

Select, ‘Tie All 1D’ to tie the X axes of all open 1D spectra. Note that in the tied mode the cross-hair cursors of each spectrum are also tied together so that they move together when you scroll in a spectrum. Select ‘OK’ and the spectra will be tied as in the following:

In the above view the three DEPT experiments are tied together. This enables the user to align common peaks between the different 1D spectra and identify the multiplicities in the Carbon data. Also this view mode can be used for setting the common spectral reference between DEPT and Carbon data. One can also perform 1D peak picking and overlay the picked peaks on any related spectrum for comparison in this multiple spectral view.

Setting Reference and Appropriate Threshold

Set the DEPT-45 '1r' file as active by selecting 'Set Active Viewport' from the Display menu, and then select ‘Peaks Table – 1D’ from the Display menu to display a blank 1D peaks table. Next, select ‘Load Table’ from the 1D Peaks Table, and select the c13.pks from the Paclitaxel directory. This will overlay the ¹³C peaks on the DEPT-45 spectrum. If the second peak from the right in the DEPT-45 spectrum matches with the peak symbol (+) of the ¹³C peak at 15.0268 ppm, then the reference is already set, and you can skip ahead to ‘Peak Picking of DEPT-45 Spectrum.’

However, if the DEPT-45 peak top is shifted from the ¹³C reference peak, then select ‘Set Reference’ from the Edit Menu, and place the cursor on the DEPT-45 peak. Then, keeping the left mouse button pressed (a circle around a ‘+’ symbol will appear) drag the cursor and release it at the center of the peak symbol (+) of the ¹³C peak at 15.0268 ppm. After releasing the left mouse button, the following dialog box will appear:

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

Open the DEPT-45 '1r' file as a single spectrum and perform peak picking. Set an appropriate threshold (e.g., 8.183e+07), and then select ‘Pick Peak Automatically - 1D’ from the Analysis menu. The program will pick 25 peaks. Next, sort the DEPT-45 peaks in the peaks table (descending order of X Value). Save the peaks as ‘dept45.pks’.

Peak Picking of DEPT-90 and DEPT-135 Spectra

Open the DEPT-90 1r file, and then set an appropriate threshold (e.g., 2.154e+07), and select ‘Pick Peak Automatically - 1D’ from the Analysis menu. The program will pick 15 peaks. Sort and save the peaks as ‘dept90.pks’.

Open the DEPT-135 '1r' file, set threshold (e.g., 3.417e+07), and then select ‘Pick Peak Automatically - 1D’ from the Analysis menu. Remember to check the ‘Negative Peaks’ option, since there are negative peaks in DEPT-135. The program will pick 23 peaks. Make sure to manually add the shoulder peak at 35.84 ppm. Sort and save the peaks as ‘dept135.pks’.

I-5. Analysis of HMQC Spectrum

The 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

Open the HMQC spectrum by selecting ‘Open Spectrum’ from the File menu, and the spectrum will be displayed along with its ‘Threshold’ control palette as shown below:

The ‘Threshold’ palette contains the following features:

Auto Redraw (updates any changes made to the threshold palette immediately. Note: use only with small data sets that are sub-matrices of large 2D spectra, since it will slow down the refresh rate)

Threshold (defines threshold by using the slider. Note: the first contour level corresponds to the threshold value)

Separation (specifies the factor by which the base threshold is multiplied to determine the cutoff of each contour level)

Number of Levels (number of contour levels is by default 20 and can be modified in the *.ini files)

Starting Level (allows stepping through the available contour levels to resolve partially overlapped peaks and to locate peak tops)

On the ‘Threshold’ control palette, uncheck ‘Auto Redraw’, set the ‘Threshold’ as 4.749e+06, set the ‘Separation’ as 1.2, set the ‘Number of Levels’ as 20 and the ‘Starting Level’ as 1. Then, click ‘Update’ and the contours will be regenerated with the new values.

Setting Spectral Reference

SpecMan’s ‘Associate Reference Spectra’ option is useful for aligning 1D and 2D peaks, by allowing simultaneous display of 1D reference spectra on a 2D spectrum. Select ‘Associate Reference Spectra’ from the Display menu, and this will bring up the following:

Check the '1D Reference Spectra along X Axis' box and then select the accompanying 'Browse' button to select the ¹H ‘1r’ file along the X-axis (F2 dimension).

Check the '1D Reference Spectra along Y Axis' and then select the accompanying 'Browse' button to select the ¹³C ‘1r’ file along the Y-axis (F1 dimension). Check the '1D Reference Peak List along Y Axis' and then select the accompanying 'Browse' button to select the ‘13C.pks’ list along the Y-axis (F1 dimension). Additional details regarding this dialog box can be viewed by selecting the Help button from the dialog box.

After selecting the appropriate reference spectra and peaks table, click ‘OK’ to display the 1D reference spectrum and the grid lines drawn at the coordinates of the 1D ¹³C peaks (these will be used to verify the peak picking results later) as shown below:

To set reference on the HMQC spectrum zoom on a well-resolved cross peak (for example, the cross peak at the lower left corner of the 2D spectrum). Check to see 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 the HMQC spectrum.

To do so, select ‘Set Reference’ from the Edit Menu. Place the cross-hair cursor on the intersection of the HMQC cross peak. Then, keeping the left mouse button pressed (a circle around a ‘+’ symbol will appear), drag the cursor and release it at the intersection of the 1H and 13C peak coordinates. After releasing the left mouse button, the following dialog box will appear:

Click ‘OK’ and the new X and Y reference ppm values for that location will be accepted.

Cross Checking 1D Peaks with 2D to Identify and Add Missing Peaks

Working with grid lines created from 1D chemical shifts has many advantages. They provide a nice way to verify 2D peak picking results, and they also identify missing 1D peaks by comparison with 2D cross peaks. For example, in this spectrum, there is no corresponding grid line (i.e. 1D ¹³C chemical shift) for the two HMQC peaks at about (H1: 4.25, C13: 76.78). By checking the DEPT-45, it can be seen that a ¹³C peak is hidden by a strong solvent peak of CHCl₃. Therefore, the ¹³C missing peak needs to be added.

To add a peak, first load the ¹³C spectrum by selecting 'Open Spectrum' from the File menu. Next load the 'dept45.pks' file into the 1D Peaks Table by selecting, 'Load Table' from the 1D Peaks Table. The DEPT-45 peaks will now be displayed on the ¹³C spectrum. The chemical shift of the ¹³C peak buried under one of the solvent peaks is seen at 76.6756 ppm. So, next load the 'c13.pks' file into the 1D Peaks Table, and select 'Add Peaks Manually - Without Refine' from the Analysis menu. Move the cursor around the solvent peak until the chemical shift displayed on the status bar reads 76.6756 ppm, and then click the left mouse button to add a peak at that location. Finally, sort the peak list in the descending order of ¹³C chemical shifts and re-save the new peak list as 'c13.pks'.

Correcting Chemical Shift Reference Offset between 1D and 2D

It is now important to verify the alignment of all 1D and 2D peak coordinates. Since the lower left peak has already been aligned, zoom on the HMQC cross peak at the upper right corner of the spectrum, and check to see if both the ¹H and ¹³C peaks are aligned well with the center of this HMBC cross peak. If there is a discrepancy, then the sweep width along X or Y needs to be adjusted.

To correct for sweep width, select ‘Spectral Parameters’ from the Edit Menu. Enter 7.9700 for the X Sweep Width as shown below:

Then, click ‘OK’ to save the spectral parameters (SpecMan saves the modified parameters so that the parameters will be retained the next time the spectrum is accessed).

Auto Peak Picking of Cross Peak Multiplets

To begin peak picking of the HMQC data, select ‘Pick Peaks Automatically – 2D’ from the Analysis menu. Make sure that the ‘Pick 2D Peaks’ dialog box is filled out as follows:

The ‘Merge Peak Multiplets’ option is utilized to pick the center of mass for cross peak multiplets, and can be performed in three separate modes:

Average

Weighted Average

Highest Peak

Using the ‘Merge Peak Multiplets’ with the ‘Weighted Average’ option, a multiplet will be merged and its center of mass will be used for the peak position. SpecMan’s peak picking algorithm uses peak width filters to discriminate noise from real peaks, and multiplets from independent peaks. These peak width filters are defined in terms of minimum and maximum box sizes for the search algorithm. For example, a peak with a width smaller than the minimum box size will be filtered out as a noise peak, while a cluster of peaks falling within the maximum box size will be merged as a multiplet.

If the user prefers to set these limits graphically, the user can select the ‘Set Graphically’ button for Peak Width Filter. This will momentarily close the ‘Pick 2D Peaks’ dialog box and the user then needs to draw a rectangular box (with the left mouse button) around a noise peak to set the minimum limit and around a cross peak for setting the maximum limit.

However, for this HMQC example, the limits are known, and 0.03 and 0.3 are used as Minimum X and Minimum Y filters for filtering noise peaks, and 0.08 and 1.0 are used as Maximum X and Maximum Y filters for merging multiplets. After the dialog box has been filled in properly, click ‘OK’ and 26 peaks will be picked and displayed in the ‘2D Peaks Table’ as shown below:

For efficient structure elucidation, it is sometimes necessary to refine the automatic peak picking results. To analyze its peak picking results, SpecMan allows the user to examine each 2D peak (one at a time) in a zoomed mode. To do this, zoom in on a certain peak of interest (so that it is the only peak visible in the spectral window), and then click on Peak #1 in the ‘2D Peaks Table.’ This will then display Peak #1 at the same level of zoom as the initial zoomed peak. Now examine the zoomed peak and compare its center with the intersection of the grid lines to make sure that the peak has been correctly picked. Then, use your keyboard’s up and down arrow keys to step through and examine the remaining peaks in the ‘2D Peaks Table.’

In this HMQC spectrum, a few odd cross peaks that exceeded the maximum box size have been missed and therefore, need to be added manually by selecting ‘Add Peaks Manually’ from the Analysis menu with the pull-right option ‘Without Refine.’ Then, move the cursor to the region of cross peaks (¹H: 2.33, ¹³C: 35.94) and click the left mouse button to add 2 new peaks. The locations of these peaks are deduced from examination of the COSY spectrum. The down-field cross peaks that arise from the phenyl groups are not carefully cleaned here because they are going to be ignored by NMR-SAMS during the partial structure elucidation process (however, several other peaks are corrected - please load the hmqc.pks file supplied with the sample data if you are having problems cleaning the HMQC peaks). Finally, 28 HMBC peaks are obtained.

Sorting and Saving Peaks, Extracting ¹H Chemical Shifts

Click the ‘Edit Table’ button from the ‘2D Peaks Table.’ The following ‘Edit Peaks Table’ dialog box will appear:

Check the following options:

‘Sort Table Entries’

‘Descending’

‘Y Value’ (¹³C chemical shifts)

‘Renumber ID's’

‘Remove Redundant Peaks’

‘Extract Coordinates to 1D Table -X’

This last option (Extract Coordinates to 1D Table – X) will extract the ¹H peak information along the X dimension from the HMQC spectrum. Click ‘OK’ and 28 ¹H peaks will be displayed in the 1D Peaks Table. Sort the ¹H peaks in the 1D Peaks Table by selecting ‘Sort Table Entries,’ ‘Descending - X Value’, and ‘Renumber Table ID's.’ Then, select ‘Save’ from the 1D Peaks Table and save the peaks as ‘h1.pks.’ Also, save the 2D HMQC peaks as ‘hmqc.pks.’

Now that the ¹H chemical shifts have been extracted from the HMQC spectrum, they can be displayed as grid lines on the HMQC spectrum. To do this, select ‘Associate Reference Spectra’ from the Display menu, click the browse button following the 1D Reference Peak List along X, and choose ‘h1.pks.’ Click ‘OK’ and the ¹H grid lines will be displayed along with the previously associated ¹³C grid lines, as shown below:

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 peak list (h1.pks) extracted from the HMQC spectral data does not include any information from the protons attached to heteroatoms. If the user wants to manually add this information, the user should open and display the ¹H spectrum, and load the ¹H peaks file by selecting ‘Load Table’ from the 1D Peaks Table (choose ‘Peaks Tables - 1D’ from the Analysis menu if the 1D Peaks Table is not already open). The ¹H peaks will then be displayed on the ¹H spectrum.

The unlabelled peaks (at 7.0, 3.58, 2.45, and 1.74 ppm) that correspond to the protons attached to heteroatoms can be added by selecting ‘Add Peaks Manually - Without Refine’ from the Analysis menu. Then, move the cursor to the location of an unlabelled peak, and click the left mouse button to add a new peak at that location. Repeat this step until all unlabelled peaks have been added. Reselect ‘Add Peaks Manually - Without Refine’ from the Analysis menu to deactivate this option. The expanded peaks table now contains 32 peaks, and is sorted and re-saved as ‘h1.pks.’

I-6. Analysis of DQF-COSY Spectrum

Setting Spectral Reference and Threshold

Open the DQF-COSY (‘COSY’) spectral file ‘1r’ from the COSY folder and set the following ‘Threshold’ palette controls:

‘Threshold’ = 2.730e+06

‘Separation’ = 1.3

‘Number of Levels’ = 20

The spectrum will appear as follows:

It is recommended to use a fairly low threshold so that all of the weak COSY peaks will appear. It is important that all of the COSY peaks, including the very weak peaks are picked, since NMR-SAMS uses the negative information from COSY. For example, two proton-bearing carbons can be forbidden to connect if their protons show no COSY peaks.

Grid Intelligence-based Peak Picking

Select ‘Associate Reference Spectra’ from the Display menu to display the ¹H reference spectrum and the ¹H peaks list (h1.pks) along both the X-axis and the Y-axis. Next, the spectral reference is calibrated in the same manner as the HMQC spectrum by checking the alignment of the 1D reference peaks with the 2D peaks. The spectral width of the F1 dimension is adjusted from 8.2636 to 8.2550 to get a better match between the 1D and F1 ¹H chemical shifts.

The COSY spectrum peaks are picked using 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 filters in the search algorithm for peak picking. After a multiplet is merged and its center of mass is calculated, SpecMan attempts to locate a grid intersection point within the specified minimum peak width box size (determined by the user). If such a grid intersection point is found, the peak center is retained as a real peak. Otherwise, it is rejected.

To pick the COSY peaks using grid intelligence-based peak picking, select ‘Pick Peaks Automatically - 2D’ from the Analysis menu. This will bring up the ‘Pick 2D Peaks’ dialog box. Check the following options:

‘Positive Peaks’

‘Negative Peaks’

‘Grid Intelligence’

Grid Distance Filter’s Minimum X PPM = 0.08

Grid Distance Filter’s Minimum Y PPM = 0.08

‘Merge Peak Multiplets’ = Weighted Average

Peak Width Filter’s Minimum X and Y PPM = 0.001 (filters for noise peaks)

Peak Width Filter’s Maximum X and Y PPM = 0.15 (filters for considering a cross-peak as a multiplet)

Next, click ‘OK’ and 49 peaks will be picked in less than 1 minute.

With regards to analysis of the peak picking results, the display of the reference 1D spectra as well as the grid lines on the 2D spectrum facilitates the user’s verification of the peak picking results.

It is not necessary to discard the COSY diagonal peaks, since NMR-SAMS will automatically disregard them later. In addition, NMR-SAMS will also merge symmetry-related peaks for all homonuclear spectra, including COSY. Although it is not crucial to pick both sets of symmetric peaks around the diagonal, SpecMan does pick both sets of symmetric peaks since it can be beneficial if peaks on one side of the diagonal are cleaner than the symmetric peaks on the other side of the diagonal. In cases where multiple grid line intersections 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 it is. NMR-SAMS will automatically include the different possible correlations to 1D peaks, and treat the cross peak as an ambiguous correlation information.

In this COSY example, some noise peaks are removed, some ignored peaks are added (for example, the peak around 2.47, 4.40), and some incorrectly picked peaks that are located away from grid line intersections are corrected (the cross peak at about 2.48, 4.41). Finally, 50 peaks are retained (please note that it you have any trouble cleaning the COSY peaks, please load the ‘cosy.pks’ file from the sample data into the 2D Peaks Table to see which peaks were removed, added, or modified). The 2D Peaks Table is sorted and renumbered using the ‘Edit Table’ option, and then saved as ‘cosy.pks’ using the ‘Save Table’ option.

Please note that the peak picking results depend upon chemical shift reference and alignment between the 1D and 2D spectra (when grid intelligence is used), in addition to the parameters in the ‘Pick 2D Peaks’ dialog box. If any of these dependencies are modified, the user may get slightly different results, but this should not drastically affect NMR-SAMS’ subsequent structure elucidation.

I-7. Analysis of HMBC Spectrum

Setting Spectral Reference and Threshold

Open the HMBC spectral file ‘1r’ from the HMBC folder and set the following ‘Threshold’ palette controls:

‘Threshold’ = 7.639e+06

‘Separation’ = 1.2

‘Number of Levels’ = 20

The spectrum will appear as follows:

Select ‘Associate Reference Spectra’ from the Display menu to display the ¹H reference spectrum and the ¹H peaks list (h1.pks) along the X-axis and the ¹³C reference spectrum and the ¹³C peaks list (c13.pks) along the Y-axis. Next, the spectral reference is calibrated in the same manner as the HMQC spectrum by checking the alignment of the 1D reference peaks with the 2D peaks. The spectral width of both dimensions is adjusted to get a better match between the 1D and 2D ¹H and ¹³C chemical shifts.

Grid Intelligence-based Peak Picking

Similar to the DQF-COSY spectrum, the peaks of HMBC are picked utilizing SpecMan’s grid intelligence-based option. To pick the HMBC peaks using grid intelligence-based peak picking, select ‘Pick Peaks Automatically - 2D’ from the Analysis menu. This will bring up the ‘Pick 2D Peaks’ dialog box. Check the following options:

‘Positive Peaks’

‘Grid Intelligence’

Grid Distance Filter’s Minimum X PPM = 0.02

Grid Distance Filter’s Minimum Y PPM = 0.5

‘Merge Peak Multiplets’ = Weighted Average

Peak Width Filter’s Minimum X PPM = 0.001 Minimum Y PPM = 0.02

Peak Width Filter’s Maximum X = 0.1 Maximum Y PPM = 2.0

Next, click ‘OK’ and 98 peaks will be picked in less than 1 minute.

As in other spectra, the display of the reference 1D spectra as well as the grid lines on the 2D spectrum facilitates the user’s verification of the peak picking results. In addition, certain areas of the spectrum can be looked at in more detail. For example, in the t₁-ridge area, the ‘Starting Level’ value in the ‘Threshold’ palette can be increased to get a better display of the real peaks. In cases where multiple grid line intersections are close to a peak and it is difficult to resolve as an unambiguous correlation, it is best to leave the picked peak as it is. NMR-SAMS will automatically include the different possible correlations to 1D peaks, and treat the cross peak as an ambiguous correlation information. Finally, 100 peaks are retained (Please note that it you have any trouble cleaning the HMBC peaks, please load the ‘hmbc.pks’ file from the sample data into the 2D Peaks Table to see which peaks were removed, added, or modified). The 2D Peaks Table is sorted and renumbered using the ‘Edit Table’ option, and then resaved as ‘hmbc.pks’ using the ‘Save Table’ option.

I-8. Editing Peak Tables before Using NMR-SAMS

In both the ¹H and ¹³C spectra, some of the resonances due to aromatic atoms are very difficult to resolve. However, the three phenyl groups in Paclitaxel can be easily identified from other spectral data (e.g. IR, UV). In situations like this, the user can ignore the three phenyl groups and let NMR-SAMS use only the well-resolved spectral data for the core structure. This is called ‘partial structure elucidation’ and will be demonstrated in this example.

Part II: Computer-Assisted Structure Elucidation with NMR-SAMS

II-1. Introduction

The first step in NMR-SAMS is to convert the ¹H, ¹³C, DEPT-45, DEPT-90, DEPT-135, DQF-COSY, HMQC and HMBC peak tables from SpecMan into correlation information for NMR-SAMS. 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, and based on this information, a set of structural building blocks are generated and an atom-atom connection matrix (ACMX) is set up. Finally, possible 2D structures are generated. If complete 2D structures are not obtained during structure elucidation, then NMR-SAMS reports only the largest possible partial structure with resonance assignments.

During the 2D structure generation process, NMR-SAMS also provides complete resonance assignments consistent with NMR data for the candidate 2D structures. Alternatively, NMR-SAMS can provide resonance assignments for 2D structures that are proposed as possible target structures by the user.

II-2. Getting Started with NMR-SAMS

Make sure to be in the …/Spectrum2001/NMR-SAMS directory, and type 'nmrsams24' at the UNIX prompt to run NMR-SAMS. Once the program has been initiated, a main NMR-SAMS window appears, as well as a ‘Status Window’ that lists the current status of the structure elucidation process and also the next possible steps.

For this part of the tutorial, use the SpecMan-generated peak lists located in the following directory: …/Spectrum2001/Data/NMR-SAMS/Paclitaxel.

II-3. Opening New Working Data Set

Select ‘New’ from the File menu, and an ‘Open File’ dialog box will appear. Click on the ‘taxol.mdf’ file and then modify its root name in the ‘File name’ text box to ‘taxoltest.mdf’ as shown below:

Then, click ‘OK’ and NMR-SAMS will create the following files in the current directory:

taxoltest.mdf – master data file. Stores all of the intermediate and final results.

taxoltest.par – default parameter file. Stores all of the control parameters used for data interpretation and structure generation. NMR-SAMS uses a set of default control parameters, but also provides the user the option of changing these control parameters (To do so, select ‘Parameters - NMR Interpretation, Setting up ACMX or 2D Structure Generation’ from the Edit menu).

taxoltest.nmr – NMR data file. Stores all of the SpecMan-converted peaks table data. The user can edit this file by selecting, ‘NMR Data File’ from the Edit menu.

taxoltest.log – log file. Stores all of the warning and error messages produced during the analysis. The user can view the log file by selecting ‘Log File’ from the Edit menu.

taxoltest.str – Stores the connection table of the generated structures and their resonance assignments.

taxoltest.lock – Prevents the data set from being opened simultaneously by two users.

Next, enter the molecular formula of Paclitaxel (C47H51NO14) into the ‘Input Molecular Formula’ dialog box as shown below:

Then click, ‘OK’ and the molecular formula is automatically interpreted for element composition and common valences. This information is written into the taxoltest.mdf file after the keyword, ‘Atoms…’. If an atom has an unusual valence, specify the valence after the element symbol (C47H51N(V)O14) where N(V) indicates a nitrogen atom with a valence of 5). Otherwise, the common valence will be adopted.

Please note that if the molecular formula is unknown, the user can enter ‘unknown’ (please see section II-17 for instructions regarding structure elucidation with an unknown molecular formula).

II-4. Conversion of SpecMan ¹H Peak List

Next, select ‘Create NMR Data File - H-1’ from the File menu, and NMR-SAMS will display a ‘Convert SpecMan H-1 Peaks Table’ dialog box. Select the ‘h1.pks’ file created in SpecMan as shown below:

Click ‘OK’ and the following dialog box will appear showing that 32 ¹H peaks were converted and written into the taxoltest.nmr file:

Click ‘OK’ and the following information dialog box will appear asking the user to supply the multiplicity information for the ¹H peaks.

Click ‘OK’ to accept this message. Note that NMR-SAMS recognizes only the following multiplet options: singlet (s), doublet (d), triplet (t), and quartet (q). All other multiplicities are entered either as unknown (u) or multiplet (m). Multiplet information will be used to eliminate inappropriate bonds during structure generation.

Select ‘NMR Data File’ from the Edit menu to modify the information obtained from the ¹H peaks in the ‘paclitaxeltest.nmr’ file (by default, the file will be edited using 'jot editor', but the user can change this to 'vi editor' by modifying the nmrsams.ini file). Edit this file by replacing the unknown multiplicity ‘u’ of 8 peaks (10, 23, 26, 28, 29, 30, 31, and 32) with singlet multiplicity ‘s’, and 7 peaks (9, 13, 17, 18, 19, 20, and 22) with doublet multiplicity ‘d’, and the remaining ones are defined either as ‘m’ (for general multiplet) or ‘u’ (for unknown), as shown in Table I [DYD1] :

Table I. The ¹H peak list of Paclitaxel

H1: …/Spectrum2001/NMR-SAMS/Paclitaxel/h1.pks

#1. 8.139 u ;1

#2. 7.747 u ;2

#3. 7.590 u ;3

#4. 7.551 u ;4

#5. 7.485 u ;5

#6. 7.478 u ;6

#7. 7.445 u ;7

#8. 7.363 u ;8

#9. 7.001 d ;9

#10. 6.285 s ;10

#11. 6.240 m ;11

#12. 5.796 m ;12

#13. 5.682 d ;13

#14. 4.946 m ;14

#15. 4.793 m ;15

#16. 4.403 m ;16

#17. 4.302 d ;17

#18. 4.207 d ;18

#19. 3.812 d ;19

#20. 3.584 d ;20

#21. 2.547 m ;21

#22. 2.453 d ;22

#23. 2.391 s ;23

#24. 2.361 m ;24

#25. 2.298 m ;25

#26. 2.236 s ;26

#27. 1.887 m ;27

#28. 1.801 s ;28

#29. 1.737 s ;29

#30. 1.695 s ;30

#31. 1.248 s ;31

#32. 1.152 s ;32

The line beginning with ‘H1’ indicates the start of ¹H peak list. The entries in the following lines indicate Peak ID, chemical shift, multiplicity, and comments (optional) for each ¹H peak. In this table, the number in the comment field corresponds to the ID of the peak in the SpecMan peaks table. Comments are not used by NMR-SAMS.

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

Next, select ‘Create NMR Data File – C13 and DEPT’ from the File menu, and NMR-SAMS will display a ‘Convert SpecMan C13 and DEPT Peaks Table’ dialog box. Select ‘Browse’ to locate the ‘c13.pks,’ ‘dept90.pks,’ and dept135.pks’ files created in SpecMan as shown below:

The default value of 0.2 is used for ‘Tolerance Along X’. Click ‘OK’ and the following message box will warn of an inconsistency detected for ¹³C peak #15 (due to the unresolved aromatic peaks which will be ignored during the subsequent analysis). Click ‘OK’ to ignore the inconsistency (NMR-SAMS will treat the multiplicity of that peak as ‘unknown’) and the following message box will appear:

Click ‘OK’ again, and the following dialog box will appear showing that 39 ¹³C peaks were converted and written into the taxoltest.nmr file:

Click ‘OK’ and the following information dialog box will appear saying that by comparison with the molecular formula, there are fewer ¹³C peaks than expected (since the molecular formula has 47 Carbons):

Since partial structure elucidation (based on the well-resolved portion of the spectral data) will be performed on this Paclitaxel example, click ‘OK’ to ignore the warning message, and the following message warning of the ¹³C peaks with unknown multiplicity will appear:

Click ‘OK’ to ignore the message, and then select ‘NMR Data File’ from the Edit menu to modify the information obtained from the ¹³C, DEPT-90 and DEPT-135 peaks in the ‘taxoltest.nmr’ file. The changes to the 13C list are displayed in Table II:

Table II. The ¹³C peak list of Paclitaxel

C13: …/Spectrum2001/Data/NMR-SAMS/Paclitaxel/c13.pks

#1. 203.826 s ;1

#2. 172.921 s ;2

#3. 171.427 s ;3

#4. 170.595 s ;4

#5. 167.252 s ;5

#6. 167.237 s ;6

#7. 142.202 s ;7

#8. 138.258 s ;8

#9. 133.914 d ;9

#10. 133.467 s ;10

#11. 132.157 d ;11

#12. 130.432 d ;12

#13. 129.415 s ;13

#14. 129.246 d ;14

#15. 128.938 u ;15

#16. 128.907 q ;16

#17. 128.568 d ;17

#18. 127.274 d ;18

#19. 84.646 d ;19

#20. 81.410 s ;20

#21. 79.331 s ;21

#22. 76.681 t ;22

#23. 75.803 d ;23

#24. 75.233 d ;24

#25. 73.477 d ;25

#26. 72.583 d ;26

#27. 72.398 d ;27

#28. 58.872 s ;28

#29. 55.282 d ;29

#30. 45.900 d ;30

#31. 43.404 s ;31

#32. 35.948 t ;32

#33. 35.855 t ;33

#34. 27.105 q ;34

#35. 22.837 q ;35

#36. 22.021 q ;36

#37. 21.035 q ;37

#38. 15.027 q ;38

#39. 9.773 q ;39

The line beginning with ‘C13’ indicates the start of ¹³C peak list. The entries in the following lines indicate Peak ID, chemical shift, multiplicity, and comments (optional) for each ¹³C peak. In this table, the number in the comment field corresponds to the ID of the peak in the SpecMan peaks table. Comments are not used by NMR-SAMS.

II-6. Conversion of SpecMan COSY Peak List

Next, select ‘Create NMR Data File – COSY’ from the File menu, and NMR-SAMS will display a ‘Convert SpecMan COSY Peaks Table’ dialog box. Select ‘Browse’ to locate the ‘cosy.pks’ file created in SpecMan as shown below:

The default value of 0.005 is used for ‘Tolerance Along X’ and ‘Tolerance Along Y’. Click ‘OK’, and the following dialog box will appear showing that 17 COSY peaks were converted and written into the taxoltest.nmr file:

The user is also notified that since NMR-SAMS assumes that all peaks are short-range couplings with unknown J-coupling constants, the user needs to denote some of the potential long-range coupled peaks as "weak" ones. 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.

Click ‘OK’ to accept this message, and then select ‘NMR Data File’ from the Edit menu to modify the information obtained from the COSY peaks in the ‘taxoltest.nmr’ file. Three DQF-COSY peaks are identified as potential long-range couplings (peaks 5 and 17 are identified as potential long-range couplings from the singlet ¹H peaks with which they interact, and peak 15 is identified as a potential long-range coupling due to its very weak peak intensity). Modify these three peaks as ‘weak’ by modifying their intensity level to 1.

(Note: a potential long-range coupled 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 peaks due to potential long-range coupling. Geminal couplings are always automatically detected by the program.)

Table III. The COSY peak list of paclitaxel

COSY: …/Spectrum2001/Data/NMR-SAMS/Paclitaxel/cosy.pks

#1. (5 - 6) 3 0.00 ;3+5

#2. (9 - 12) 3 0.00 ;8+14

#3. (11 - 24) 3 0.00 ;10

#4. (11 - 25) 3 0.00 ;11

#5. (11 - 28) 1 0.00 ;12+46

#6. (12 - 15) 3 0.00 ;16+23

#7. (13 - 19) 3 0.00 ;18+33

#8. (14 - 21) 3 0.00 ;20+39

#9. (14 - 27) 3 0.00 ;21+43

#10. (15 - 20) 3 0.00 ;24+35

#11. (16 - 21) 3 0.00 ;27+37

#12. (16 - 22) 3 0.00 ;25+41

#13. (16 - 27) 3 0.00 ;28+42

#14. (17 - 18) 3 0.00 ;30+31

#15. (17 - 19) 1 0.00 ;29

#16. (21 - 27) 3 0.00 ;40+44

#17. (31 - 32) 1 0.00 ;47+49

The entries in the lines above indicate Cross Peak ID, ID’s of the correlated ¹H peaks (in parenthesis; for ambiguous correlations the ID’s of all possible ¹H peak correlations are included), peak intensity levels (which are classified as four types; strong (3), medium (2), weak (1), and unknown (0) – the default value is 3), J-coupling (optional, the default value is 0), and comments (optional, with a maximum size of 80 characters), for each COSY cross peak.

For short-range coupled DQF-COSY peaks, intensity levels should be either 3 or 2. For long-range coupled DQF-COSY peaks, the intensity level should be 1. If an intensity level of 0 is used, the program will expect actual J-coupling values in the field that represents J-coupling. One or more spaces are used as a delimiter for all items, except for 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 ID’s of the peaks in the SpecMan peaks table. For merged peaks, these numbers are shown with a + sign. Comments are not currently used by NMR- SAMS.

II-7. Conversion of SpecMan HMQC Peak List.

Next, select ‘Create NMR Data File – HMQC’ from the File menu, and NMR-SAMS will display a ‘Convert SpecMan HMQC Peaks Table’ dialog box. Select ‘Browse’ to locate the ‘hmqc.pks’ file created in SpecMan as shown below:

The default value of 0.01 is used for ‘Tolerance Along X’ and the ‘Tolerance Along Y’ value is 0.2.

Click ‘OK’ and the following warning box will appear because the peak picking results in the aromatic region have not been edited:

As already stated, the aromatic groups will be ignored during the process of structure elucidation. Click ‘OK to All’ to ignore all similar messages and the following dialog box will appear:

Click ‘OK’ to ignore the message and the following message warning about the ¹H peaks for which no HMQC cross peaks were observed will be displayed:

Click ‘OK’ to accept this warning message, and 27 HMQC peaks are converted and written into the taxoltest.nmr file as shown in Table IV:

(Please note that unlike other 2D peaks where ambiguous correlations are allowed, HMQC peaks must have exactly two correlated 1D peaks).

Table IV. The HMQC peak list of Paclitaxel

HMQC: …/Spectrum2001/Data/NMR-SAMS/Paclitaxel/hmqc.pks

#1. (9 - 3) ;1

#2. (11 - 5) ;2

#3. (12 - 1) ;3

#4. (15 - 4) ;5

#5. (15 - 8) ;6

#6. (16 - 7) ;4

#7. (18 - 6) ;7

#8. (19 - 14) ;9

#9. (22 - 17) ;11

#10. (22 - 18) ;10

#11. (23 - 10) ;12

#12. (24 - 13) ;13

#13. (25 - 15) ;14

#14. (26 - 11) ;15

#15. (27 - 16) ;16

#16. (29 - 12) ;17

#17. (30 - 19) ;18

#18. (32 - 24) ;20

#19. (32 - 25) ;19

#20. (33 - 21) ;21

#21. (33 - 27) ;22

#22. (34 - 31) ;23

#23. (35 - 23) ;24

#24. (36 - 32) ;25

#25. (37 - 26) ;26

#26. (38 - 28) ;27

#27. (39 - 30) ;28

The line beginning with ‘HMQC’ indicates the start of HMQC peak 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, ID’s of the correlated ¹³C & ¹H peaks shown in parenthesis, and comments (optional, with a maximum size of 80 characters), for each HMQC cross peak. One or more spaces are used as a delimiter for all items except comments which are separated by “;”. Comments are not currently used by NMR-SAMS.

II-8. Conversion of SpecMan HMBC Peak List

Next, select ‘Create NMR Data File – HMBC’ from the File menu, and NMR-SAMS will display a ‘Convert SpecMan HMBC Peaks Table’ dialog box. Select ‘Browse’ to locate the ‘hmbc.pks’ file created in SpecMan as shown below:

The default value of 0.005 is used for ‘Tolerance Along X’ and the default value of 0.08 is used for ‘Tolerance Along Y’. Click ‘OK’ and the following warning box will appear indicating that a cross peak picked by SpecMan will be discarded because its ¹H or ¹³C chemical shift does not match a 1D ¹H or ¹³C peak, within the specified tolerance:

Click ‘OK to All’ to accept all similar messages, and the following warning message will appear indicating that ambiguous cross peaks have been obtained:

Click ‘OK to All’ and 95 HMBC peaks are converted and written into the taxoltest.nmr file as shown in Table V:

(Please note that it can be seen that some peaks have ambiguous correlation to 1D peaks due to very close chemical shifts. 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).

Table V. The HMBC peak list of Paclitaxel

HMBC: …/Spectrum2001/Data/NMR-SAMS/Paclitaxel/hmbc.pks

#1. (1 - 10) 3 ;2

#2. (1 - 19) 3 ;1

#3. (1 - 30) 3 ;3

#4. (2 - 12) 3 ;6

#5. (2 - 15) 3 ;7

#6. (2 - 20) 3 ;5

#7. (3 - 10) 3 ;8

#8. (3 - 26) 3 ;9

#9. (4 - 23) 3 ;10

#10. (5 6 - 1) 3 ;13

#11. (5 6 - 2) 3 ;15

#12. (5 6 - 9) 3 ;11

#13. (5 6 - 12) 3 ;12

#14. (5 6 - 13) 3 ;14

#15. (7 - 10) 3 ;16

#16. (7 - 11) 3 ;17

#17. (7 - 24) 3 ;20

#18. (7 - 25) 3 ;19

#19. (7 - 28) 3 ;18

#20. (8 - 12) 3 ;21

#21. (8 - 15) 3 ;22

#22. (9 - 1) 3 ;23

#23. (10 - 5) 3 ;27

#24. (10 - 10) 3 ;24

#25. (10 - 11) 3 ;28

#26. (10 - 28) 3 ;29

#27. (10 - 31) 3 ;26

#28. (10 - 32) 3 ;25

#29. (11 - 2) 3 ;30

#30. (13 - 4) 3 ;31

#31. (15 16 - 8) 3 ;33+36

#32. (15 16 - 31) 3 ;32+37

#33. (15 16 - 32) 3 ;34+35

#34. (17 - 8) 3 ;38

#35. (18 - 2) 3 ;40

#36. (18 - 5) 3 ;41

#37. (18 - 12) 3 ;39

#38. (19 - 17) 3 ;46

#39. (19 - 18) 3 ;42

#40. (19 - 21) 3 ;44

#41. (19 - 22) 3 ;43

#42. (19 - 27) 3 ;45

#43. (20 - 14) 3 ;49

#44. (20 - 17) 3 ;48

#45. (20 - 18) 3 ;52

#46. (20 - 19) 3 ;47

#47. (20 - 21) 3 ;50

#48. (20 - 27) 3 ;51

#49. (21 - 13) 3 ;57

#50. (21 - 19) 3 ;59

#51. (21 - 24) 3 ;55

#52. (21 - 25) 3 ;53

#53. (21 - 29) 3 ;54

#54. (21 - 31) 3 ;58

#55. (21 - 32) 3 ;56

#56. (22 - 19) 3 ;60

#57. (24 - 19) 3 ;61

#58. (24 - 24) 3 ;64

#59. (24 - 25) 3 ;62

#60. (24 - 29) 3 ;63

#61. (25 - 12) 3 ;66

#62. (26 - 25) 3 ;67

#63. (26 - 28) 3 ;68

#64. (27 - 19) 3 ;73

#65. (27 - 21) 3 ;72

#66. (27 - 22) 3 ;69

#67. (27 - 27) 3 ;71

#68. (27 - 30) 3 ;70

#69. (28 - 13) 3 ;74

#70. (28 - 19) 3 ;75

#71. (28 - 21) 3 ;77

#72. (28 - 30) 3 ;76

#73. (29 - 5) 3 ;80

#74. (29 - 6) 3 ;82

#75. (29 - 9) 3 ;78

#76. (29 - 15) 3 ;79

#77. (29 - 20) 3 ;81

#78. (30 - 13) 3 ;83

#79. (30 - 16) 3 ;86

#80. (30 - 17) 3 ;84

#81. (30 - 18) 3 ;85

#82. (30 - 30) 3 ;87

#83. (31 - 10) 3 ;91

#84. (31 - 24) 3 ;89

#85. (31 - 29) 3 ;88

#86. (31 - 31) 3 ;92

#87. (31 - 32) 3 ;90

#88. (32 33 - 13) 3 ;93

#89. (32 33 - 22) 3 ;94

#90. (32 33 - 29) 3 ;95

#91. (33 - 16) 3 ;96

#92. (34 - 32) 3 ;97

#93. (36 - 31) 3 ;98

#94. (39 - 16) 3 ;100

#95. (39 - 19) 3 ;99

The line beginning with ‘HMBC’ indicates the start of HMBC peak 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, ID’s of the correlated ¹³C and ¹H peaks shown in parenthesis, and comments (optional, with a maximum size of 80 characters), for each HMBC cross peak. One or more spaces are used as a delimiter for all items except comments which are separated by “;”. Comments are not currently used by NMR-SAMS.

II-9. Generation of Building Blocks

Once the SpecMan-generated peak lists have been converted, it is time to generate Building Blocks. During this step, NMR-SAMS interprets the molecular formula, ¹³C, ¹H, and HMQC spectral data (all data except ¹³C spectral data is optional), and generates sets of possible building blocks for structure generation. To do this, select ‘Building Blocks’ from the Analysis menu and the following dialog box will appear:

Select ‘OK to All’ to assume a default range of 0 to 3 for the protons attached to the #15 ¹³C peak and all similar peaks, and the following dialog box will appear:

NMR-SAMS compares the number of constituent carbon atoms to determine the symmetry of the molecule and warns the user that the number of ¹³C peaks is fewer than the number of constituent carbon atoms (47) in the molecule and that partial structure elucidation will be performed (NMR-SAMS only considers carbon atoms labeled by the available ¹³C peaks). Click ‘OK’ to accept partial structure elucidation and the following dialog box will appear:

NMR-SAMS warns the user that some exchangeable protons are not observes as cross peaks in the HMQC spectrum and tries to assign them based on chemical shifts to heteroatoms. Since ¹H peak #2 (7.747ppm) is due to an aromatic proton, the user cannot accept this, so click ‘No’ to assign the ¹H peaks one by one, and the following will appear:

Type ‘unknown’ for proton #2 and then click ‘OK.’ For the remaining protons (#9, #20, #22, and #29), accept the suggested values by selecting ‘OK’, and the following summary will be displayed for the 4 assigned proton peaks:

Click ‘OK’ and the building blocks will be displayed:

Building block #15 (CH?) has an uncertain number of attached protons because its ¹³C multiplicity is unknown, and building blocks #40 - #47 are also represented as ‘CH?’ because there were not any ¹³C peaks observed (due to peak overlap). These building blocks (15, 40-47) correspond to the aromatic carbons in the Paclitaxel molecule, but because their spectral properties are not available or incomplete, they will be ignored (displayed in red) during the structure generation process.

The results of the building blocks process are written into the *.mdf file after the keyword ‘Atoms.’ Please note that for each step, NMR-SAMS keeps only one copy of the results in the master data file (*.mdf). When any step is repeated, the previous results of that step and any previous steps will be overwritten.

To modify the appearance of the building blocks, the user can select ‘Display Options’ with the following pull-right choices: Balls, Carbon Symbols, Numbers, Chemical Shifts, Protons, Molecular Formula, etc. from the Display menu.

II-10. User-Defined Building Blocks

The user can edit the heteroatoms building blocks (there are restrictions on editing Carbon building blocks) that have been generated by NMR-SAMS. This is useful in designating the carbon atoms that are part of the aromatic building blocks and in excluding these building blocks from the subsequent structure generation process. To do so, select ‘User-Defined Building Blocks’ from the Analysis menu and the following palette will appear:

From their chemical shifts, building blocks #8, #9, #11 –14 and #16 - 18 are also recognized as aromatic, and therefore, select ‘Modify’ and ‘Ignored Atom’ from the ‘User-Defined Building Blocks’ palette. Next, click on building block #8 and it will be changed to red and treated as an ignored atom. Continue to click on the rest of the building blocks that need to be ignored (#9, #11 –14 and #16 – 18) and then select ‘OK.’ In this way, all of the aromatic atoms are defined as ignored atoms, and will be excluded from the structure generation process, as shown below:

II-11. Interpretation of Bond Constraints

To interpret all available 2D spectral data as bond constraints, select ‘Bond Constraints’ from the Analysis menu. In this step the various bond constraints, e.g. the H-H BC’s from COSY and the C-H ones from HMBC, are transformed into C-C bond constraints based on the HMQC-derived C-H connectivity. The following ‘Summary of Constraints’ dialog box will appear:

Click ‘OK’ and the following building blocks with fixed bonds will be displayed:

Atoms with unsatisfied valences are marked by an asterisk (*), and are displayed in a different color (blue by default). Fixed bonds with unknown bond types are displayed as dashed lines, and can become single, double, or triple bonds in the structure generation process.

Note that NMR-SAMS automatically adds a carbonyl and 5 carboxylic groups based on the ¹³C chemical shifts. For the Paclitaxel data this is correct, but sometimes the automatically added functional groups may not be correct. In those cases, the user can modify them by selecting ‘User-Defined Bond Constraints’ from the Analysis menu.

NMR-SAMS also provides the user with the ability to interact with the atoms in the NMR-SAMS display window and the bond constraints listed in the Connection Table (shown below):

If the user clicks on a bond constraint in the Connection Table, the relevant atoms in the NMR-SAMS display window will be highlighted. Conversely, if the user clicks an atom in the NMR-SAMS display window, the relevant entries in the Connection Table will be highlighted.

II-12. User-Defined Bond Constraints

Once the bond constraints have been generated, it is important to review the building blocks, the fixed bonds, and the available bond constraints. If the user has a priori knowledge about the structure, the user can define additional constraints by selecting ‘User-defined Bond Constraints’ from the Analysis menu and the following dialog box will appear:

In the Paclitaxel molecule, the user knows that based on their ¹³C chemical shifts (ranging from 172.921 - 167.237 ppm), carbons 2 – 6 are either ester or amide groups. In the HMBC spectrum, a peak is observed between C5 or C6 (because they are partly overlapped) and the NH at ¹³C: 167.2, ¹H: 7.0. This means that either C5 or C6 can be an amide group, so arbitrarily C6 is set as an amide group. To do this, select ‘Delete’ from the User-Defined Bond Constraints’ dialog box and then click on C6 and O59 to delete the bond between them. Then, select ‘Add’ and ‘Single Bond’ and click on C6 and N48 to add a single bond between these two atoms.

Finally, click ‘OK’ to accept the newly created functional groups and the ‘Summary of Constraints’ dialog box will appear again:

Select ‘OK’ and the updated functional groups and fixed bonds will be displayed as shown below:

Typically when insufficient spectral data is available, structure generation generally takes more time than usual to converge. Therefore, the user is urged to input any additional user-defined bond constraints to improve the convergence and the quality of the structure generation.

II-13. 2D Structure Generation

Once the user has given NMR-SAMS all available spectral information (plus any user-defined information) for structure generation, the user needs to modify the default parameters for the 2D structure generation process.

One of the major bottlenecks in computer-assisted structure elucidation is the efficiency of structure generation (a factor of: computational time and the quality and number of candidate structures generated). NMR-SAMS’ structure generator searches all plausible 2D structures consistent with the data, and if the spectral data is precise and has fewer ambiguities, then NMR-SAMS will generate the correct, unique structure within a reasonable computation time. However, as the size of the molecule and the number of free bonds increases (molecules with sizes >30 heavy atoms), the structure elucidation process becomes more and more time consuming. NMR-SAMS applies several heuristic rules to speed up this process, and the user has some control over these rules.

To do so, select ‘Edit Parameters – 2D Structure Generation’ from the Edit menu and the following will appear:

Since there is an HMBC peak at ¹H: 2.453, ¹³C: 87.71 that corresponds to a four-bond coupling between OH50 and C9 (whereas all other HMBC peaks correspond to either two-bond or three-bond couplings), modify the ‘Maximum Limit for Bond Constraint Violation’ from ‘0’ to ‘1.’ The remaining parameters are left with their default values. Click ‘OK’ to apply the modified parameter.

Next, select ‘Generate 2D Structures’ from the Analysis menu, and the following dialog box will appear:

The user is prompted to define a range of ‘dummy bonds’ to be added to the generated structures/sub-structures. For partial structure elucidation, NMR-SAMS’ structure generator will try to generate the largest substructure that is consistent with the available data. In some instances, a dummy bond is used to satisfy a free bond by assuming that it is connected to an ignored atom.

Type ‘3 3’ to add exactly three dummy bonds to the generated structures (Paclitaxel’s three phenyl groups will not be included in the structure elucidation process), and click ‘OK.’

The structure generation process will be initiated, and the following dialog box will be displayed:

This dialog box shows the progress of the structure generation process. At any time the user can abort the structure generation process by clicking ‘Stop’ (it will take a few seconds to stop the structure generation).

In approximately 2 minutes, the following dialog box will appear to announce that 12 complete structures (and 38 partial structures) have been generated:

Click, ‘Yes’ to save the 12 complete structures and the 38 partial structures in ‘taxoltest.str’ and the following dialog box displaying a summary of the structure generation results will appear:

Click ‘OK’ and the first structure will be displayed as shown below:

A structure browser is displayed so that the user can look through the remaining 11 candidate structures and the 38 partial structures. The 12 candidate structures have three dummy bonds (denoted as ‘~’) that indicate connections to ignored atoms. Although these candidate structures are chemically incomplete, they are considered to be ‘complete structures’ because they do not have any remaining free valences. The partial structures are listed in descending order of the number of generated bonds.

Note: If the user had only connected C5 and C6 to an oxygen by a double bond (because it was not apparent which one was an amide or an ester group) while entering the user-defined bond constraints, then the structure generation would have taken about 2 hours to complete, and 24 candidate structures would have been generated. Only 12 of these are unique structures, since each duplicate pair of candidate structures shows the exchange of C5 and C6. It can be seen that most of the multiple candidates arise from the lack of HMBC connectivity between C2 and H3 (on C16), as well as between C4 and C10 (both are quaternary carbons).

II-14. Editing Generated Structures

After the structures have been generated, the user can further edit the structures to include the atoms that were ignored during the structure elucidation process. For example, display structure #1 and then select ‘Generated Structures’ from the Edit menu to display the following molecular editor:

The user should build three phenyl groups using the ‘Add’, ‘Bond’ and ‘Single’ options from the molecular editor (build three phenyl groups with alternating single and double bonds). Then, connect the three phenyl groups to the three carbons that have dummy bonds (‘~’) and select ‘OK’ to close the molecular editor. Next, select ‘Display Options – Refine’ from the Display menu and the structure will be refined and displayed as shown below:

Please note that when the user edits a generated structure, NMR-SAMS will not check the edited structures against bond constraints, chemical shift information, etc.

II-15. Exporting NMR Data, Resonance Assignments and Structures

NMR-SAMS enables the user to export chemical shift correlations. Select ‘Export – Chemical Shift Correlation’ from the File menu, and the chemical shift correlations will be written into a ‘taxoltest.spc’ file.

To export the results of resonance assignment, select ‘Export – Assignment’ from the File menu, and the ¹³C and ¹H assignments of all the atoms in the selected structure will be written into a ‘taxoltestx.rst,’ where x is the number of the structure. 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 will be included to enable the resolving of ambiguous NOE peaks, and identification of through-space NOE connectivities.

To export structures into a structure file (*.mol, *.mdl or *.sdf), select ‘Export – Structures’ from the File menu and this will bring up the following dialog box:

Select either *.mol, *.mdl or *.sdf and then click ‘OK’ and the structure will be exported into ‘taxoltest001.mol’, ‘taxoltest001.mdl’ or ‘taxoltest001.sdf.’

The structure elucidation process of Paclitaxel is now complete.

II-16. Report Generation

Now that the structure generation process has been completed, it is important to be able to create detailed reports as efficiently as possible. SpecMan and NMR-SAMS contain convenient clipboard functions for report generation. In SpecMan, open the Paclitaxel molecule by selecting ‘Open Molecule’ from the Edit menu. Select the ‘taxoltest001.mol’ file from the …/Spectrum2001/Data/NMR-SAMS/Paclitaxel/Paclitaxel-MF folder, and the following molecule will be displayed:

SpecMan allows the user to renumber the molecule, since the user may want to use a different numbering system than that of the program. To renumber a molecule, select, ‘Molecule’ from the Edit menu. This will bring up the following Molecular Editor palette:

To renumber an atom, select ‘Renumber’ from the Molecular Editor dialog box. This feature will allow you to renumber atoms in a structure to conform to IUPAC or other conventions. The Molecular Editor dialog box will appear as shown below:

Select the number that you would like to begin renumbering with (for example, 1), and type it in the text box next to ‘Renumber.’ Then click on the atom in the molecule that you would like to renumber as atom 1 (for example, atom 33). Internally, atom 33 will now become atom 1, and the old atom 1 will be renumbered as atom 33. Additionally, the assignments will also be swapped so that they remain with the correct atom, and the assignment table will be updated with the new atom numbers. As you click on atoms, the program will increment the initial renumbering number. For example, the next atoms will be labeled two, three and so on. If you had begun your renumbering with 20, then the next atoms would be labeled 21, 22, 23, etc. The renumbering of atom 33 is shown in the following:

Comparison of the initial molecule (shown on Page 57) with the above-modified molecule shows the old atom number 33 now renumbered as atom 1.

Once the molecule has been renumbered, select ‘Copy Molecule – Active Molecule’ from SpecMan’s Edit menu. The molecule will be saved as a molecule.gif file, and can be viewed using any graphics program that reads *.gif files. The molecule.gif file can then be imported to a PC and the picture can be pasted into a word document (as shown below):

If the user has opened up multiple molecules, all molecules in the molecular window can be copied. To do so, select ‘Copy Molecule – Molecule Window’ from the Edit menu. The molecule will be saved as a molwindow.gif file

The user can also copy the assignment table, by selecting ‘Copy – Assignment Table’ from the Edit menu. The assignment table will be saved as an assignment.txt file and can be viewed using a screen editor, or the assignment.txt file can be imported to a PC and the table can be pasted into a word document (as shown below):

The assignment table will appear in text format in the Word document. In order to convert the assignment table into actual table format, highlight the text portion of the assignment table, and under Word's Table menu, select Convert Text to Table.

Similarly, the user can copy spectra and 1D and 2D Peaks Tables and paste them all into Word documents for easy report generation.

II-17. Structure Elucidation With Unknown Molecular Formula

NMR-SAMS enables the user who does not have a high resolution molecular formula, but has molecular weight information, to perform structure elucidation.

The procedure for structure elucidation with an unknown molecular formula is very similar to the procedure for a known molecular formula (utilize the tx-nomf folder under …/Spectrum2001/Data/NMR-SAMS/Paclitaxel). The differences are as follows:

1. When selecting ‘Create NMR Data File’ from the File menu, input the molecular formula as ‘UNKNOWN’ (see II-3).

2. During the peaks table conversion (see II-4 through II-8), the program will not check the results against a molecular formula, so some warning messages will not appear.

3. During generation of building blocks, NMR-SAMS will generate building blocks according to the observed ¹³C peaks. For this example, fewer building blocks will be generated compared to those obtained in section II-9, as shown below:

4. During the ‘User-Defined Building Blocks’ step (section II-10), the user has to add more ignored building blocks that correspond to phenyl groups. To do this, select ‘User-Defined Building Blocks’ from the Analysis menu. The following palette will be displayed:

First, modify building blocks #8, #9 and #11 – 18 as ignored atoms. Then, add 11 oxygen atoms by selecting ‘Add’, ‘O’ for the element, ‘0’ for the proton count, and ‘2’ for the valence and then click 11 times in the main graphics window.

Add 8 carbon atoms (for which ¹³C peaks were not observed) by selecting ‘Add’, ‘C’ for the element (ignored atom is automatically selected), ‘unknown’ for the proton count, and ‘4’ for the valence, as shown below:

Click 8 times in the main graphics window, select ‘OK’ to accept the added atoms, and NMR-SAMS will rearrange the building blocks and set up the ACMX again (the resulting building blocks should be the same as the final results described in II-10).

For the remainder of the structure elucidation process, the operations and results are exactly the same as those described for the example with a known molecular formula.

Computer-Assisted Structure Elucidation of Paclitaxel (Taxol) Using SpecMan and NMR-SAMS

Table of Contents

Computer-Assisted Structure Elucidation of Paclitaxel (Taxol) Using SpecMan and NMR-SAMS

Separation (specifies the factor by which the base threshold is multiplied to determine the cutoff of each contour level)

Number of Levels (number of contour levels is by default 20 and can be modified in the *.ini files)

Computer-Assisted Structure Elucidation of Paclitaxel (Taxol)
Using SpecMan and NMR-SAMS

Computer-Assisted Structure Elucidation of Paclitaxel (Taxol)
Using SpecMan and NMR-SAMS