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

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

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

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

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

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

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

I-1. Transferring Processed Spectrum from NMR Spectrometers

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

H-1/

C-13/

DEPT/

DQFCOSY/

HMQC/

HMBC/

NOESY/,

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

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

I-2. Analysis of 1D Proton Spectrum

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

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

Setting Reference

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

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

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

I-3 Analysis of 1D Carbon Spectrum.

Setting Reference and Appropriate Threshold

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

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

Automatic Peak Picking

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

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

Manually Removing and Adding Peaks

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

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

Sorting, Editing and Saving Peaks Table

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

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

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

I-4 Analysis of DEPT Spectra

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

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

Setting Reference and Appropriate Threshold

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

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

Peak Picking of DEPT-45 Spectrum

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

Peak Picking of DEPT-90 and DEPT-135 Spectra

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

I-5 Analysis of HMQC Spectrum.

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

Setting Threshold

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

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

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

Setting Spectral Reference

Next, set spectral reference. On 2D spectra, SpecMan’s Associate Reference Spectra Option is useful for aligning 1D and 2D peaks. To use this feature, select the Associate Reference Spectra option in the Display menu. When this option is selected, a dialog box appears (as seen

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: 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 Spectra

I-5 Analysis of HMQC Spectrum.

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

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