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Computer-Assisted Structure Elucidation of Q-2
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 betulinic acid (Q-2) 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 step-by-step instructions for each task in SpecMan, while Parts II describes step-by-step instructions for each task in NMR-SAMS.

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

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

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

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 Spectra from NMR Spectrometers

The spectral data of Q-2 was acquired on a Varian Unity-plus 720 MHz spectrometer and processed (FT, phase correction, etc.) with VNMR 5.1. The concentration of the sample used was 0.084 M in pyridine-d₅. In order to import Varian processed spectra into SpecMan, the user needs to transfer the phasefile and the procpar files for each spectrum to the working directory on the PC.

For this tutorial, the sample data is located in the Data directory on the CD. Copy the Data.zip file from the Data directory into the Spectrum2001 directory on your PC. Then extract the zip file using an extraction program, such as WinZip. The default location for the sample data is: C:\Spectrum2001\Data\SpecMan\Q-2 for SpecMan data, and C:\Spectrum2001\Data\NMR-SAMS\Q-2 for NMR-SAMS data. The following subdirectories will appear under the C:\Spectrum2001\Data\SpecMan\Q-2 folder:

H-1

C-13

DEPT

COSY

HMQC

HMBC

NOESY

I-2. Analysis of 1D Proton Spectrum

From the Program Manager or the Start Menu, click the SpecMan icon in the Spectrum2001 group to launch the SpecMan program. Then, open a 1D ¹H file by selecting ‘Open Spectrum’ from the File menu. Select the File Type as Varian, and then use the file browser to change to the H-1 directory and double click on the phasefile in this directory as shown below:

After double clicking on 'phasefile', 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 procpar file, but the user may want to change the spectral reference.

In this case, zoom on the small, weak peak due to residual g-proton of C₅D₅Nat about 7.55 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) 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 7.55, then type ‘7.55’ 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 procpar file.

In this case, 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, open the ‘phasefile’ 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 middle peak of pyridine at 135.5 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 spectrum when the cursor is moved to the 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 9.906e-003 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.’ Thirty-eight 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 (triplets between 150 to 123 ppm; a total of nine peaks, numbered 28 through 36) 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 solvent peak entries will be removed from the 1D Peaks Table. Next, re-click on the ‘Remove Peaks’ button from the Analysis menu to deactivate the option.

SpecMan also gives the user the ability to add peaks 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 desired position to add a new peak. Please note that there are not any new peaks added to the ¹³C spectrum (We assume that the user has not yet identified the overlapping peak at 16.44 ppm. This will be pointed out later by NMR-SAMS and the peak will be split into two peaks).

Sorting, Editing and Saving Peaks Table

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 29 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 Spectra

The DEPT experiment usually consists of DEPT-45, DEPT-90, and DEPT-135. When a Varian spectrometer is used, the DEPT experiment is stored in the form of arrayed 1D experiments. SpecMan automatically detects the multiple 1D spectra and displays a slider in the following 1D Control Panel:

In order to view each experiment type, the user can move the slider on the 1D Control Panel to position 1 for DEPT-45, to position 2 for DEPT-90, and to position 4 for DEPT-135, or the user can view all three experiments 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 DEPT ‘phasefile’ from the DEPT folder. To add additional spectra, re-select ‘Add’, and then select the same DEPT ‘phasefile’ two more times (this is necessary since the DEPT experiment is saved as an arrayed spectrum). 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. The following warning dialog box will appear:

Click ‘OK’ and the following multiple spectra will be displayed:

To display the DEPT-45, DEPT-90 and DEPT-135 spectra, set the top spectrum as active (select ‘Set Active Viewport’ from the Display menu and then click on the top spectrum). Since DEPT-45 is the first position on the slider, it is already at the right location. Next, set the second spectrum as active and move the slider to the second position to display DEPT-90. Then, set the third spectrum as active and move the slider to the fourth position to display DEPT-135.

In order to keep the three DEPT experiments separate, rename the spectral titles by choosing ‘Set Labels’ from the Edit menu and rename the spectra as ‘DEPT 45,’ ‘DEPT 90,’ and ‘DEPT 135’, respectively.

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

Open the DEPT ‘phasefile’ as a single spectrum, and leave at the first position for DEPT-45. 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 Q-2 directory. This will overlay the ¹³C peaks on the DEPT-45 spectrum. If the first peak from the left in the DEPT-45 spectrum matches with the peak symbol (+) of the ¹³C peak at 109.9134 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 109.9134 ppm. After releasing the left mouse button, the following dialog box will appear:

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

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

Overview:

Part I: NMR Analysis and Assignment with SpecMan

I-1. Transferring Processed Spectra from NMR Spectrometers

I-2. Analysis of 1D Proton Spectrum

I-3. Analysis of 1D Carbon Spectrum

I-4. Analysis of DEPT Spectra

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

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