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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:

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

Overview:

Part I: NMR Analysis and Assignment with SpecMan

I-1. Transferring Processed Spectrum from NMR Spectrometers

I-2. Analysis of 1D Proton Spectrum

I-3. Analysis of 1D Carbon Spectrum

I-4. Analysis of DEPT Spectrum

I-5. Analysis of HMQC Spectrum

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