Spectrum Research, LLC.
CHORDS Tutorial
Unix Version
Table of Contents
Step 2- Importation of a processed spectrum.
Step 3- Selection of a cross peak for analysis
Step 4- Saving the selected region in a sub-matrix
Step5 -Working with the selected cross peak
Step 6- Preparation of Input files for simulation
Step 7- Preparation of the INP file
Step 8- Preparation of the J file
Step 9- Preparation of the XFB file
Step 10- Using J-slider for Simulation
Step 11- Comparing Experiment with Simulation
CHORDSTM
Tutorial
This manual describes the 2.0 release of the Windows 95/98/2000 and Windows NT 4.x version of CHORDSTM.
Copyright Notice
Copyright © 1996 through 2001 Spectrum Research, LLC. All rights reserved.
No part of this document may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into any language in any form by any means without the written permission of Spectrum Research, LLC.
Spectrum Research, LLC. reserves the right to change the information in this document without prior notice.
Trademarks
CHORDSTM is a trademark of Spectrum Research, LLC.
Acknowledgments
CHORDS has been developed by Drs. R. Ajay Kumar and A. Majumdar in the laboratories of Profs. Hosur and Govil at the TATA Institute of Fundamental Research, India. Portions of CHORDS are copyright 1990 through 1994 TATA Institute of Fundamental Research, India, and are exclusively licensed to Spectrum Research, LLC. Title and full ownership rights to the converted/modified CHORDS remains solely with Spectrum Research, LLC., and CHORDS is asserted to be Spectrum Research’s proprietary information and trade secret.
Credits
If the results (figures and/or data) obtained by CHORDSTM are used for publication purposes, please refer to them in the following manner or any other equivalent form:
"CHORDSTM software, developed by Spectrum Research, LLC., was used to compute the results in this publication".
Estimation of J(H, H) in Paclitaxel (Taxol)
This example shows the procedure of using CHORDS to estimate the J(H2’, H3'), J(OH2'-H2'), and J(NH-H3') values of the cross peak at (5.79, 4.79 ppm) in the DQF-COSY spectrum of Paclitaxel (taxol). The DQF-COSY spectrum was collected on Bruker DMX 300 MHz spectrometer. The 2rr, procs, and proc2s files were transferred via ftp to use as import to CHORDS. In the following example CHORDS has been used to study a spin subsystem with two passive spins 2’OH and NH in addition to the active spins 2’H and 3’H as shown in figure 1.
Fig.1 Spin subsystem of a cross peak in DQFCOSY spectrum of taxol.
Type chords at the UNIX prompt to launch Chords program.
Step 2- Importation of a processed spectrum.
Select File/Import & Export to import the Bruker file. An Import a File dialog box is displayed. Select Bruker as the Vendor Type. Click Browse button of Input Filename to select the 2rr file of Paclitaxel. Next click Browse button of Output Filename to specify an output filename in the appropriate directory (e.g. taxol-all in this example).
Click Show Spectral Parameters to see the parameters. A parameter dialog box (as shown below) appears with the parameters read from the procs and proc2s files of the 2rr data. If the parameters are right, click OK to accept or else change the parameters and click OK. Turn off the option Set Cross Peak Limits to import the entire spectrum. Turn on the option Load into Chords after Import. Click OK. After the import, the DQF-COSY spectrum is saved in a file taxol-all.exp and also displayed in the graphics window. This file will be used by CHORDS to estimate the J-coupling constants.
Step 3- Selection of a cross peak for analysis
Next turn the cross-hair cursor on by selecting Analysis/Crosshair Cursor. Zoom into the cross peak of 2’H and 3’H (around w2= 5.79, w1=4.79 ppm). Press the right mouse button and drag the mouse, until the displayed rectangular zoom box covers the desired cross peak and release the right mouse button. The selected spectral region will be zoomed in. The right mouse button can be used in this manner to perform continuous zooms. You can also select Analysis/Zoom to define the zoom region, but this allows only one level of zoom for each selection of this option. Other related commands, such as Analysis/Previous Zoom or Analysis/Reset Zoom, can be used to switch to the previous zoom or the full view of the spectrum.
Step 4- Saving the selected region in a sub-matrix
After the cross peak is properly zoomed, select File/Save Submatrix, and type a filename to save this cross peak as a submatrix, (e.g. taxol-2-3 for this example). The cross peak is saved in a file taxol-2-3.exp.
Step5 -Working with the selected cross peak
Select File/Open to open the submatrix taxol-2-3.exp. If necessary, adjust the Threshold, Separation, or Number of Levels in the Threshold Palette and click Update to get a better display of the cross peak. (For this example, using threshold 3.143e+06, separation 1.2, and number of levels as 20 gets a good display).
Turn on the cross hair cursor by selecting Analysis/Crosshair Cursor. Select Display/Unit/Hertz to change the units as Hertz. Move the cursor to the center of the peak and note down the coordinates: w2:1739.2 Hz, w1: 1438.9 Hz.
Step 6- Preparation of Input files for simulation
Before performing a simulation, you must prepare the following three files (.inp, .j and .xfb files) as described below. CHORDS uses these files along with the .exp file containing the selected cross peak for simulation and J-coupling measurements. Among the set of CHORDS generated files, the .exp, .sim (which is the simulation data) and .con files (which has saved contours) are binary files, and the rest (.inp, .j, and .xfb) are all text files.
Step 7- Preparation of the INP file
Select File/Prepare Input/Create INP File. A dialog box as shown below appears. In this dialog box default values are used for all the options, except Type of Experiment, which is selected as “Double quantum-filtered COSY”.
Click Input Chemical Shifts at the bottom of the dialog box, this displays an Input Chemical Shifts dialog box as shown below. Type 4 for Number of Spins. Next, Type 1739.6 for Chemical Shift of "Proton" 1 (which is the 3’H), click Next to accept it.
Type 1440.1 for Chemical Shift of Proton 2 (i.e., 2’H), click Next to accept it.
Type 1075.8 for Chemical Shift of Proton 3, (i.e., 2’OH), click Next to accept it.
Type 2101.5 for Chemical Shift of Proton 4, (i.e., NH), click Next to accept it.
Note down the sequence of the protons that you input in this step, since it determines the order to input the J-coupling constants in the following step.
Step 8- Preparation of the J file
Select File/Prepare Input/Create J File. A Create J File dialog box is displayed (as shown below). In this dialog box default values are used for all options except the ones described below.
Input the data of all possible combinations of the coupled protons in the following sequence (this sequence is determined by the order in which the chemical shifts were entered in step 7, for the 3’H, 2’H, 2’OH, and NH protons):
3’H-2’H, 3’H-2’OH, 3’-NH, 2’H-2’OH, 2’H-NH, and 2’OH-NH.
Type 3’H-2’H (this label will be used to denote the J-coupling for the J slider, see step 9) for the First Proton Couple. Turn on the button labeled "Coupled", if it is not already on. Type in appropriate values for the minimum, maximum, and initial J-coupling constant between them. (e.g. 0, 12, 3). Click Next to accept it.
Next type 3’H-2’OH for the Proton Couple 2. Turn off the button labeled "Coupled". Click Next to accept it.
Next type 3’H-NH for the Proton Couple 3. Turn on the button labeled “Coupled”. Type in appropriate values for the minimum, maximum, and initial J-coupling constant between them (e.g. 0, 12, 9). Click Next to accept it.
Next type 2’H-2’OH for the Proton Couple 4. Type in appropriate values for the minimum, maximum, and initial J-coupling constant between them (e.g. 0, 12, 5). Click Next to accept it.
Next type 2’H-NH for the Proton Couple 5. Turn off the button labeled “Coupled”. Click Next to accept it.
Next type 2’OH-NH for the Proton Couple 6. Leave the "Coupled" button off. Click Next to accept it.
After the expected number of coupling constants (4 for this example) has been accepted, the program prompts a dialog box (as shown below) to confirm it. Click No to terminate adding coupling constants. The initial setting of J values will be saved. If you made a mistake in any of the entries, repeat the above steps to correct the entries.
Step 9- Preparation of the XFB file
Select File/Prepare Input/Create XFB File. A Create an XFB File dialog box is displayed. Enter 6 as the Line Width for both dimensions. Enter 1739.2 and 1438.9 as the Offset for w2 and w1, respectively., The default values of Digital Resolution, Time Domain Size and Frequency Domain Size values are automatically calculated based on the submatrix. Retain the default values for these entries. Select the appropriate Window Function (e.g. Sinebell for both dimensions), and type in the appropriate Window Function Shift (e.g. 0 for both dimensions). Next click OK to save these values.
After the three input files (.j, .xfb, and .inp files) are prepared, a J Values palette is displayed (see below), showing the current J values for each pair of Coupled Protons. If this is not on, select Display/J Values. If this toggle is on and you still don't see the palette, it means there are errors in your input files. Repeat the above steps (7 to 9) to check the input files.
Step 10- Using J-slider for Simulation
Next click Simulate in the J Values palette to simulate the spectrum. The experiment and simulated spectra will be displayed side by side (left-experiment and right-simulation). Adjust the display of the spectra, by changing Threshold, Separation, or Number of Levels in their corresponding Threshold palette and click Update to apply the changes.
Click and drag the sliders in the J Values palette to adjust the J values. Click Simulate and the new J values will be used for the next simulation. Repeat this step until the two multiplets look very similar.
Step 11- Comparing Experiment with Simulation
To compare with a better view, select Display/Overlay/Without Offset to overlay the simulation on top of the experimental peak. For finer adjustment of J values, click the slider, then press the up or down arrow key to increase or decrease the J value in steps of 0.1 Hz.
Also use the zoom functions to get a better view of the overlaid multiplets. To measure the distance separation between the two mutliplets, first click the left mouse button at one point, then keep it pressed and drag the mouse to the other point. As you are dragging, the status bar below the graphics window (where the cursor locations are usually displayed) will display in real time the distance separation between the two points along both dimensions. The display units can be changed using Display/Units.
For this example, a good fitting between the simulated and experimental spectra (along both dimensions) is observed when the following J values are used:
J(3’H-2’H) = 3.0
J(3'H-NH) = 8.5, and
J(2’H-2'OH) = 5.5,
The coupling constants are thus estimated.
CHORDS enables automatic and manual peak picking. These procedures are included in the Analysis menu. The picked peaks are written into a peaks table that can be saved as text file (.pks extension) and exported to other spreadsheet style programs. After peak picking, you can use the peak annotation capabilities of CHORDS to label the peaks with Greek and other alphanumeric characters. Tools for addition and removal of peaks are also available. With the Tie Views function, you can tie the X and Y axes of the experimental spectra with the corresponding axes of the simulated spectra. This is used before overlaying the spectra. CHORDS has options for creating hard copy plots in the form of postscript files. Use the Create Postscript procedure in the file menu to generate hard copy outputs. The postscript output file has both experiment and simulation plots.
For more details regarding the usage of these functions, please refer to the on-line help in CHORDS.