| Felix Tutorials |
The Analytical module of Felix assists chemists in resonance assignment for small molecules. It includes controls for structure display, spin system detection, and manual assignment of 1D/2D peaks to atoms.
In Lesson 1: Resonance assignment of brucine, you will learn the basic steps involved in assigning a natural product using conventional 2D spectra.
This lesson presents the basic steps of an NMR spectrum assignment using prepared 1D and 2D data: the H-1, DEPT-135, TOCSY, and HMQC spectra, of brucine (MF: C23H26N2O4) and its structural diagram prepared with ISIS/Draw).
The topics covered in this lesson are:
1. Setting up for the lesson
2. Starting Felix
3. Going to the Analytical module
Select the Analytical/Project menu item from the menubar.
4. Setting up the database
This procedure typically takes several seconds.
5. Viewing the project entity through a spreadsheet
Select the Analytical/Project menu item again.
The project entity is presented in a spreadsheet, and you can browse through its fields.
Many fields contain zeros or nulls, since the project is not fully defined yet. Also note that there are twelve experiment columns, meaning that you can define twelve experiments in one project.
6. Adding the 1D H-1 experiment to the project
Two additional ways of adding experiments to a project are shown in Steps 6-9 of this lesson.
Select the Analytical/Experiment menu item. In the Add Analytical Experiment control panel, type h1 as Experiment Title and toggle Take Current to on. Select OK.
This adds the currently displayed H1 experiment to the project.
This adds the H1 experiment to the project with the current display parameters. You can change the display parameters using the Experiment/Change Attribute menu item in the Experiments table (select the Analytical/Experiments menu item to display the Experiments table).
7. Adding the DEPT-135 experiment to the project
In the OPEN FILE control panel, choose Felix New Data (*.dat) as File Type. When the list appears with names of 1D spectra in the ./ directory, select br_dept.dat (the 1D DEPT spectrum). Select OK
In the PLOT PARAMETERS control panel, leave the default values for all parameters and select OK.
The peaks are labelled on the DEPT spectrum. The C13 peaks are also listed in the spreadsheet table Peaks - pic:dept.
Select the File/Close menu item from the table to close it.
This demonstrates two ways of adding experiments to a project: you can add the currently displayed experiment or a saved experiment.
8. Repeating Step 6 for the HMQC experiment
Adjust the display of the spectrum until it is satisfactory.
The peaks are labelled on the HMQC spectrum. The HMQC peaks are also listed in the spreadsheet table Peaks-xpk:hmqc.
Select the File/Close menu item from the table to close it.
This adds the currently displayed HMQC experiment to the project.
The HMQC spectrum is added to the project.
9. Repeating Step 7 for the TOCSY spectra
Go to the Analytical Experiments spreadsheet menubar.
If it is not open, select Analytical/Experiments to open it.
Select the Experiment/Add menu item.
In the Add ANALYTICAL EXPERIMENT control panel, enter tocsy as Experiment Title and turn off the Take Current toggle. Select OK.
This opens a TOCSY spectrum.
In the OPEN FILE control panel, choose Matrix (*.mat) as File Type. When the list appears with names of 2D spectra in the ./ directory, select br_tocsy.mat (the 2D TOCSY spectrum). Select OK.
In the 2D DISPLAY PARAMETERS control panel, click the Set button if you find the display satisfactory. Otherwise you can click the Full button to display the full spectrum or the Zoom button to zoom in on a certain spectral region. If the display is still not satisfactory, click No in the GENERAL MESSAGE control panel to return to the 2D DISPLAY PARAMETERS control panel.
If the HMQC peak labels are displayed on the spectrum, ignore them for now.
This imports the TOCSY peaks.
The peaks are labelled on the TOCSY spectrum. The peaks are also listed in the spreadsheet table Peaks-xpk:tocsy.
Select the File/Close menu item from the table to close it.
10. Checking the project entity
Check the project entity after all experiments are added, as described in Step 5.
The previously zero or null fields now have values.
11. Drawing the full HMQC spectrum
Now go to the Experiments table and select the hmqc spectrum by clicking the third row and then clicking the Select Experiment icon.
Now click the Full Plot icon to draw the full HMQC spectrum.
12. Performing prototype pattern detection
Information about the current stage of prototype pattern collection is displayed in the text window. The prototype pattern collection finishes quickly, and the following information appears in the text window:
Nr of prototype patterns generated:(8)
The 2D protopattern detection took 1 seconds
Also, a spreadsheet containing the 8 prototype patterns is displayed (Protopatterns table).
13. Writing the result of prototype pattern detection into a file
Go to the Protopatterns table and select File/Save As. Set the Selection to br_protos.txt and select OK.
The text window shows information about the success of the action:
Wrote table: br:proto
Created file: ./br_protos.txt
14. Visualizing prototype patterns
The next step is to visually inspect the prototype patterns. Several ways of seeing prototype patterns are provided through the Protopatterns spreadsheet: you can draw frequencies of prototype patterns as lines on top of a contour plot, spawn tiles, or draw a strip plot.
On the HMQC spectrum, you see six lines on D1, and 12 lines on D2 if it is a full plot. Among the 12 lines on D2, only six are C13 lines, while the remaining six are the symmetrical images of the D1 H1 lines. You can recognize them based on whether they are correlated to any HMQC peaks.
You can also choose to display the TOCSY spectrum and then plot the prototype pattern on it.
Hold down the right mouse button and select the Clear Frequencies item from the menu that appears.
This clears the frequency lines on the spectrum.
15. Making a tile plot of prototype pattern
The second way to visualize prototype patterns is to spawn tile plots from them. This allows you to concentrate only on frequencies and peaks belonging to them, which are present in this prototype pattern.
Select the third prototype pattern from the Protopatterns table and click the Tile Plot icon.
If you want to change the tile plot attributes, select the Preferences/Tile Plot menu item from the table.
The tile plot is displayed.
Press <Ctrl>-c (if you were in intensity plot mode) to see the contour plot of the HMQC spectrum tiled by this prototype pattern.
If you want to switch back to intensity plot mode, press <Ctrl>-i.
You can also display frequencies by clicking the Draw icon in the table.
16. Making a strip plot of a prototype pattern
Using the tile plot functionality, you can concentrate on peaks and their immediate surroundings which belong to a prototype pattern. You can also use strip plots to see strips surrounding the frequencies in vertical or horizontal orientations.
Press <5> and use the large cross-hair cursor to pick one of the boxes.
This action (Jump) places only that small region on the screen and returns from tile plot mode.
You see the seven HMQC peaks relevant to the frequencies of the third prototype pattern displayed in horizontal strips. You can also display the frequencies by selecting the Draw icon.
The strip plot helps you verify if there are outstanding peaks that have common chemical shifts with the frequencies in this prototype pattern.
17. Copying a prototype pattern to the frequency clipboard
In practice, because of peak overlap or missing peaks, the automatically detected prototype patterns may have wrong connectivities, which need to be manually corrected. Felix provides a set of tools to verify and edit the prototype patterns. These actions can be accessed from the pullright menu by selecting Analytical/Frequency. The first step in this procedure is to copy the frequencies of a certain prototype pattern to the frequency clipboard.
In the Frequency Clipboard menu, select Copy Proto To Clipboard. In the control panel that appears, select 3 from the List of Protos and select OK.
The third prototype pattern is now copied to the clipboard list. This list can be manipulated (you can add or delete frequencies to or from the list, swap the order of two frequencies, delete duplicate frequencies, sort the list, or zero the list). You can also display the list as lines on top of the matrix plot and spawn a tile and strip plot from it.
You can see the sorted clipboard by selecting the View Clipboard item from the Frequency Clipboard menu. The result should look like this:
The Frequency Clipboard List contains the following frequencies:
# Freq(ppm) Atom
--- --------- ----
1 127.315 Y
2 64.597 Y
3 60.076 Y
4 56.463 Y
5 31.469 Y
6 26.774 Y
7 5.881 X
8 4.099 X
9 3.851 X
10 3.139 X
11 2.347 X
12 1.456 X
18. Drawing the clipboard prototype pattern
As described in Step 11, you can use the Experiment Table to display the HMQC or TOCSY spectrum before viewing the prototype pattern.
Select the Draw Clipboard item from the Frequency Clipboard menu. Click Redefine Color in the control panel that appears and define D1 and D2 Color as Blue and Green, respectively. Select OK.
The spind system is displayed on the 2D spectrum.
To clear the display of the frequencies, click the right mouse button and select Clear Frequencies in the popup menu.
Similarly, you can select Tile Clipboard or Strip Plot Clipboard menu item from the Frequency Clipboard menu to display the clipboard spin system in tile or strip view. These tools are useful when you edit the prototype pattern in the clipboard, as shown in the next step.
19. Editing the clipboard prototype patterns
If you find it necessary to edit the prototype pattern in the clipboard, choose the Add One, Delete One, Swap Two, or Remove Duplicates menu item in the Frequency Clipboard menu to add, remove, swap, or purge frequencies in the prototype pattern.
The third prototype pattern in the Protopatterns table is updated.
20. Import and display molecular structure
The prototype patterns usually give you an idea of the assignment of the frequencies to individual atoms in the molecule. Once you are ready to assign the resonances, you can input the chemical structure and interactively assign the resonances in a 1D or 2D peak table to the atoms.
Select the Analytical/Read Coordinates menu item. In the control panel, set File Type to MDL MOL files (.mol) and select file brucine.mol. Select OK.
The structure is displayed with atom labels on.
In the 1 Player control panel, select Zoom. Then drag the cursor around the structure to zoom the structure until it is satisfactory.
You can also rotate, translate, or reset the display by choosing options in the 1 Player control panel.
Since this is a planar structure (i.e., all Z coordinates are zero), you can only rotate it in the plane of the screen.
Click Exit in the 1 Player control panel to close it.
You can select the Analytical/Draw Molecule, Analytical/Label..., and Analytical/Color... menu items to redraw the molecule, change the display of labels, or change the colors if necessary.
Currently, all bonds are displayed as single lines. The carbonyl group C1=O19, double bond C1=C13, and phenyl ring consisting of C20-C25 are not displayed as such.
21. Assigning 1D DEPT peaks to atoms
Select Preference/Frame Layout... and check 2 Frames Left/Right. Select OK.
Now there are two empty frames.
If it is not open, select Analytical/Experiment... to open it.
Then click the Draw icon on the table.
The DEPT spectrum is displayed in the new frame. If the DEPT peak labels are not displayed, toggle on View/Draw Peaks.
Make sure Frame 2 is highlighted. Select Edit/Peaks to display the DEPT peaks in a Peaks-pic:dept table.
The values in the name column are all null, which means that the corresponding peaks are not assigned yet.
Now select the first peak (168.904 ppm) by highlighting the first row in the peak table.
From the chemical shift it obviously belongs to the only carbonyl carbon c1.
Select Peak/Assign One from the table and click c1 with the large crosshair cursor.
Notice that the name cell of peak 1 is now c1.
If you want to unassign a peak, highlight it and select Peak/Unassign One. The name of the peak is restored to null.
The DEPT assignments are stored in an ASCII file.
Select File/Close from the Peaks-pic:dept table to close it.
22. Assigning the HMQC peaks to atoms
You can also assign the C13 frequencies in the HMQC peak table to the carbon atoms. Note that quaternary carbons are not present in the peak table, and that since hydrogen atoms are not explicitly displayed in the structure, you cannot assign the H1 frequencies to hydrogens.
Make sure Frame 2 is highlighted. Select HMQC experiment from the Analytical Experiments table.
If it is not open, select Analytical/Experiment... to open it.
Click the Draw icon in the Analytical Experiments table to display the HMQC spectrum.
Select Edit/Peaks.
The HMQC peaks are displayed in a Peaks-xpk:hmqc table. Notice that the column NameD1 and NameD2 are all nulls, which means that the corresponding frequencies are not assigned yet.
Now highlight 127.315 (or the NameD2 cell just next to it) in the third row.
From the chemical shift it obviously belongs to the sp2 methine carbon C11. (Remember that double bonds are not displayed.)
Select Peak/Assign One from the table and click C11 with the large crosshair cursor.
You'll notice that the name cell of this frequency changes to C11.
If you want to unassign a frequency, highlight it (either the chemical shift or the name) and select Peak/Unassign One. The name of the peak is restored to null.
The HMQC assignments are stored in an ASCII file.
23. Exiting Felix
To exit Felix, select the File/Exit menu item.