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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 you through the process of Computer-assisted Structure Elucidation of the Paclitaxel (Taxol, Fig.1) molecule. This document is intended for the day-to-day users of SpecMan and NMR-SAMS and we assume that the users of this document 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.

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.

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 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 were collected on 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. The processed files from Bruker are transferred via ftp or other means to the SGI workstation. In order to import Bruker processed spectra into SpecMan, one needs to transfer the 1r/2rr files along with their corresponding procs and proc2s files to the working directory on the SGI workstation. For this example, a main working directory called Paclitaxel was created with the following sub-directories:

H-1/

C-13/

DEPT-45/

DEPT-90/

DEPT-135/

HMQC/

DQFCOSY/

HMBC/

Next, the 1r/2rr and procs/proc2s files of each NMR experiment were transferred 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/paclitaxel for SpecMan data, and /usr/share/Spectrum/Data/NMR-SAMS/paclitaxel for NMR-SAMS data. The NMR experimental data is located in the sub-directories (H-1 through NOESY). Before proceeding further please make a backup copy of the distribution data that was sent with the demo software.

I-2 Analysis of 1D Proton Spectrum

At the UNIX prompt, type specman to launch the SpecMan program. Next, Open the 1D file by selecting Open Spectrum option in the File menu. The dialog box that is shown below appears and select the File Type as Bruker, Then use the file browser to change directory to H-1 and double click on the 1r file in this directory. The 1D spectrum will be displayed in a 1D Window as seen below. The first step will be to set the reference. Although the reference parameters are obtained from the procs file, the user may want to change the spectral reference.

Setting Reference

Before setting reference, zoom the desired peak by using the right mouse button which activates a rubber-band zoom option. 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. After zooming, select the Set Reference option in the Edit Menu. Place the cross hair cursor at the top of the peak and click the left button. A Set 1D Reference dialog box appears with the X chemical shift of the current location. Type 7.27 in the X Reference PPM. Then click OK to accept the new reference. To reset the expansion to full view, select Reset Zoom (in the Display Menu) or the Reset Zoom icon in the tool bar. Once reference is set, the relevant spectral parameters will be saved with the data 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.

I-3 Analysis of 1D Carbon Spectrum.

Setting Reference and Appropriate Threshold

After opening the 1r file from the C-13 directory, set reference in a similar manner as described for 1D ¹H. The reference is set on the CHC1₃ peak at 77.22 ppm. Next do the 1D peak picking of ¹³C spectrum. Before peak picking, set the appropriate threshold. Select the Set Threshold button in the 1D Control Panel, which is displayed below. This will activate a red horizontal cursor on the 1D spectral window as the mouse is moved in this window. Move this horizontal cursor to a suitable position so that it is just above the noise level. 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 2.381e+07 for threshold.

Automatic Peak Picking

Choose the Pick Peaks Automatically from the Analysis menu, then choose 1D from the Pick Peaks Automatically pull-right menu. This activates a Pick 1D Peak dialog box shown below. You can also activate the 1D peak picking by selecting the 1D peak picking icon in the tool bar.

Turn off the Negative Peak option and then click OK. After peak picking, the 40 picked peaks are displayed on the spectrum, and are also listed in the following 1D Peaks Table.

This peaks table can be resized, and the two sections can be made larger or smaller with the small hash bar in the middle of the table. The four main peaks table operations can be performed via the buttons at the bottom of the table. Please see the online help for more information about working with peaks tables.

Manually Removing and Adding Peaks

Next, delete the three strong solvent peaks by choosing the Remove Peaks option 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 using the left mouse button (enclosing peaks 18-20) to remove the peaks. After removing the three solvent peaks (peak numbers 18-20), deactivate the Remove Peaks button in the Analysis menu by selecting it again. SpecMan also has an Undo option which allows restoring peaks that have been removed accidentally. The Undo option is activated by either selecting Undo in the Edit Menu or the second icon from the left on the tool bar.

The large peak at 167.25 PPM, is an envelope of two overlapping peaks. To see this better, first zoom into the dominant peak. Then, add a peak to this one by first selecting Add Peaks Manually from the Analysis menu and then choosing the Without Refine option from the Add Peaks Manually pull-right menu. After selecting this option, click the cursor at the place where the smaller peak can be seen affecting the shape of the dominant peak to add a peak. Next, deactivate Add Peaks Manually by selecting it again along with the pull-right menu option.

Sorting, Editing and Saving Peaks Table

Next select Edit Table button in the 1D peaks table, and a edit dialog box (shown below) appears. 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 38 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 Spectrum

In order to get the ¹³C multiplicity information, we need to use only two DEPT experiments: DEPT-90 and DEPT-135. In some instances, DEPT-45 may be used to detect potential errors such as missing peaks. For this example, we do the peak picking in all three spectra (DEPT-45, DEPT-90 and DEPT-135), but use only the DEPT-90 and DEPT-135 peak lists along with ¹³C peak list for the multiplicity analysis in NMR-SAMS.

Setting Reference and Appropriate Threshold

Similar to the ¹³C spectrum, the DEPT spectra are analyzed one at a time. First the DEPT-45 spectrum is opened and displayed for setting the reference. The second peak from the right in the spectrum is set to 15.0268 PPM 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 ¹³C sub-directory. The ¹³C peak positions will be annotated on the DEPT spectrum. This can be used as a guide to set reference on the DEPT peak. If the peak top of the second peak from the right in the DEPT-45 spectrum matches with the peak symbol (shown as “plus”) corresponding to ¹³C peak at 15.0268 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 Display 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 15.0268 PPM). After releasing the left mouse button, a dialog box (similar to the one shown in Page 9) is displayed, showing the current chemical shift. Click OK and the reference of the DEPT-45 spectrum will be the same as that of the ¹³C spectrum.

Peak Picking, Removing Peaks and Manually Adding Peaks

Next perform peak picking of DEPT-45 spectrum. Set the appropriate threshold as described before (or type 8.183e+07 in the threshold box on 1D Control panel). Select auto 1D peak picking from the Analysis menu and the program picks 25 peaks. Next sort the peaks in the peaks table in the descending order of X Value as described previously. The sorted peaks are saved as dept45.pks by using the Save command in the 1D peaks table.

Next process the DEPT-90 and DEPT-135 spectra in a similar way. Remember to first set the reference in these spectra by following the same procedure as described above for DEPT-45. For DEPT-90 use the threshold 2.154e+07 and 15 peaks will be picked by auto 1D peak picking. For DEPT-135 use the threshold 3.417e+07 and also turn the Negative Peaks button on in the 1D peak picking dialog box (because there are negative peaks in DEPT-135). 23 peaks will be picked in the DEPT-135 spectrum. Make sure to manually add the shoulder peak at 35.84 ppm. 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. Due to peak overlap in 1D proton spectrum, HMQC spectrum is used to extract 1D ¹H chemical shifts. This process is described in the next few sections.

Setting Threshold

The 2rr file 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 graphics window along with a Threshold palette (both displayed below).

The Threshold palette (shown above) 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 the Threshold as 4.749e+06, Separation as 1.2, and the Number of Levels as 20.

Setting Spectral Reference

Next set spectral reference by selecting the Associate Reference Spectrum in the Display menu. 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) is displayed along the Y-axis (F1 dimension). When this option is selected, a dialog box appears (as seen on the next page) and prompts for the file names of reference spectra and reference peaks table. 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 its corresponding peaks table, 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, which indicate the plausible locations of the 2D peaks in the HMQC spectrum. This will help the user verify the results of peak picking 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.

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

Working with grid lines created from 1D chemical shifts has many advantages. It provides a nice way to verify 2D peak picking results, and also identifies missing 1D peaks if there are any by comparing with the 2D cross peaks. For example, in this spectrum, you will notice that 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. Next add the ¹³C missing peak to the ¹³C peaks table as described below:

First load the 1D ¹³C spectrum by selecting Open Spectrum in the file menu. Next load the DEPT-45 peaks table by selecting the Load Table button in the 1D Peaks Table and choosing file dept45.pks in the sub-directory DEPT-45. The DEPT-45 peaks will be displayed on the 1D ¹³C spectrum. The chemical shift of the ¹³C peak buried under one of the solvent peaks is seen at 76.6756 ppm. Next load the ¹³C peaks table and select Add Peaks Manually with the pull-right option Without Refine in the Analysis menu. Move the cursor around the solvent peak until the chemical shift displayed on the status bar reads 76.6756ppm. At that point click the left mouse button to add a peak. Next sort the peak list in the descending order of ¹³C chemical shifts and save the new peak list in the same file c13.pks.

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 and then move the cross hair to a cross peak which is at the upper most right corner of the 2D spectrum. Check if both the ¹H and ¹³C peak are well aligned 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 display menu. The following spectral parameter dialog box appears, enter the new Sweep Widths along X or Y in this dialog box..

In the paclitaxel HMQC data the ¹H chemical shifts do not match well. There is a discrepancy of about 0.08 PPM between the 1D and 2D ¹H spectral width. After changing the Sweep Width along X from 7.9787 to 7.9700 PPM the peaks are well aligned in the 1D and 2D.

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.

Auto Peak Picking of Cross Peak Multiplets.

Peak picking of HMQC data consists of auto peak picking followed by manual editing of the peak list. SpecMan uses a novel peak picking algorithm to automatically pick the center of mass of cross peak multiplets. To start peak picking select Pick Peak Automatically in the Analysis menu with the 2D option in the Pick Peak Automatically pull-right menu. A dialog box appears with various peak picking options as shown below.

Turn off the Negative Peaks button as well as the Grid Intelligence option. 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 pick cross peaks as a merged multiplets with a center of mass. These filters are defined in terms of a minimum and maximum box size for the search algorithm. The minimum box size is used to filter noise peaks. The maximum box size corresponds to the width of an average cross peak multiplet. The limits can be determined by using the “Set Graphically” button in the Pick 2D Peaks 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 0.03 and 0.3 as Minimum X and Minimum Y for filtering noise peaks, and 0.08 and 1.0 as Maximum X and Maximum Y for merging multiplets. Next click OK to pick peaks. 26 peaks are automatically picked by SpecMan and the 2D Peak Table will be displayed as shown here.

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: Computer-Assisted 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.