The previous tutorials: CHAPTER 3: "Tutorial I: Using NMRanalyst", CHAPTER 4: "Tutorial II: Setting Analysis Parameters", and CHAPTER 5: "Tutorial III: Combining Analysis Results" cover the basic NMRanalyst spectral analysis. This tutorial covers generating a generic line list for nitrogen resonances (SECTION 6.2: "Generation of a Generic 15N Resonance List"), positioning nitrogen atoms in determined molecular carbon skeletons (SECTION 6.3: "15N HMBC Spectrum Analysis"), using additional spectra to reduce the number of possible molecular skeletons (SECTION 6.5: "Using DQF-COSY" and SECTION 6.6: "Using DQ 1,1-ADEQUATE"), and placing unobserved heteroatoms in determined structures (SECTION 6.7: "Adding Heteroatoms").
strychninedataset containing the experimental data. Delete its
N15_HMBCsubdirectories. This removes the provided NMRanalyst parameter settings for this compound. In NMRanalyst, start
[Full NMRanalyst]and deselect the
[Show All Input Fields]switch.
strychninedirectory. Select the HMBC spectrum type. Change to the 1D Analysis workwindow. Load from directory
procparand run the 1D Analysis workwindow. Load
HMBC/1d_analysis.logto add the table of obtained reference corrected numerical signal descriptions with CDCl3 solvent resonances marked for removal. Rerun the workwindow. Display the shown carbon spectrum (file
procpar. Run the analysis. Display the created and shown
F1: Start of Spectrumto
2533.119(taking into account the 6.052 Hz correction from the 1D carbon spectral analysis), and run the workwindow. Switch to the 2D Analysis workwindow. After optimization, as described in the previous tutorial, the final 2D Analysis parameters are:
[F2 Phase]switches in
-0.0020997* ppm, and
0.0548430* ppm. Run the 2D Analysis and it runs the Report workwindow. In the Graphic workwindow, specify the same phase functions as above and display the shown
structure.plotfile illustrates the detected correlations in graphical form.
F1: Start of Spectrumto
2313.039and run the FFT. Switch to the 2D Analysis workwindow. The strongest HMBC correlations result from 2-bond and 3-bond couplings. Often the 2D Analysis workwindow
Integralthreshold can be set so that a sufficient number of 2-bond and 3-bond correlations are detected without detection of longer-range correlations. But longer-range HMBC correlations are indistinguishable from 2-bond and 3-bond correlations. Set the
Integralthreshold high enough to exclude noise and spectral distortions from detection. Set this field now to
1.5, run the 2D Analysis workwindow, and it auto-runs the Report workwindow. From the Graphic workwindow, display the shown
A quality 1D proton spectrum can be acquired in seconds. But for nuclei such as carbon or nitrogen, acquiring the 1D spectrum tends to be slower than acquiring HSQC and HMBC spectra. For strychnine, the 15N HMBC spectrum was acquired without a corresponding 1D nitrogen spectrum. Similar to SECTION 5.5: "Structure Identification Without 1D Carbon Spectrum", a generic line list needs to be generated by NMRanalyst to describe the nitrogen frequencies. Select spectrum type N15_HMBC and switch to its FFT workwindow. Load file
N15_ghmbc.fid/procpar. Run the 15N HMBC spectrum transform.
To generate the generic resonance list for this nitrogen dimension, switch to the 1D Analysis workwindow. Set the
Input File Format menu to
[Generate Generic List] which hides input sections that do not apply. Set
Use Relaxation Time [s] to
0.015. Copy the four values from the FFT workwindow F1 dimension to the 1D Analysis workwindow fields of the same name:
Observe Frequency is
Spectral Width is
Start of Spectrum is
-261.032 Hz, and
Number of Points is
320. Run the 1D Analysis workwindow and the generic 15N line list consisting of 78 resonances is generated.
F1 1D Analysis Output Fileto
nitrogen.out. Due to the use of the generic 15N line list, set
Map F1 Frequencies± to
58Hz to cover the 1D Analysis 115 Hz line spacing. Set
1.5. In the Report workwindow, set
Redetermined F1 Resonance Listto
nnitrogen.out. Run the 2D Analysis and it runs the Report workwindow. From the Graphic workwindow, display the
n15_hmbc.specfile with analyzed correlation locations (
n15_hmbc_areas.plot) as shown. Determined nitrogen frequencies are saved in the specified
nnitrogen.out, so an exhaustive search of the nitrogen axis is no longer needed.
F1 1D Analysis Output Fileto
nnitrogen.outand delete the
Map F1 Frequencies± entry. In the Report workwindow delete the
Redetermined F1 Resonance Listentry to not overwrite the newly created line list. Now run the 2D Analysis again and it auto-runs the Report workwindow. Switch to the AssembleIt workwindow. Change the
1D: Nitrogenfield to
nnitrogen.outand start the workwindow. Display the created
NC_corrs.plotfile. It is a circle diagram. As only a few correlations were derived, click the NMRgraph
[Redo Layout]menu item to obtain the shown layout.
From the previous analysis results, the possible strychnine structures can be derived. Return to spectrum type HMBC (not N15_HMBC) and select the AssembleIt workwindow. Select the
COMBINE NMR ANALYSIS RESULTS section. Set
nnitrogen.out. Delete the
INADEQUATE field entries. For faster structure generation, the HMBC
Weak field should be specified. To see how to choose this value, deselect the
AssembleIt: ELUCIDATE MOLECULAR STRUCTURE FROM NMR DATA section in this input screen and start the workwindow. The middle of the output screen shows the detected 127 HMBC correlations:
HMBC 'HMBC/report.log': 499.87 MHz F1Diff=0.155026 F2Diff=0.016914 F1-Range=+/-9.74 F2-Range=+/-6.50 1: # 225 C1= 3 H2= 35 F1= 140.032 F2= 4.092 I= 20.120 2: # 889 C1= 10 H2= 34 F1= 77.391 F2= 4.090 I= 16.070 ... 127: # 770 C1= 9 H2= 10 F1= 116.005 F2= 7.198 I= 1.507
The strongest HMBC correlation has an integral of
20.120 and the weakest one an integral of
1.507. A consistent way to acquire HMBC spectra and experience in analyzing such spectra will yield insights on how to set this
Weak threshold. Set the AssembleIt workwindow HMBC
Weak field to
2.2, declaring the bottom one-sixth of these correlations to potentially contain longer-range correlations. A similar
Weak threshold could be set for N15_HMBC. As only two nitrogens were detected, the obtainable speed improvement is limited and considering all of these correlations to be potential longer-range correlations is fine.
Now select the
AssembleIt: ELUCIDATE MOLECULAR STRUCTURE FROM NMR DATA section. Typically about 20% of expected 2-bond HMBC correlations are not observed due to unfavorable 2JC,H coupling constants. Set
Evaluate: Unobserved Bonds to
10 for AssembleIt to try to derive up to this number of unobserved bonds from longer-range correlations. For aromatic systems, 3-bond correlations are typically stronger than 2-bond ones. Set the
Evaluate: option menu to
[Bonds Over sp2
] to more likely recover such bonds. Finally, set
4-Bond HMBC Correlations to
Long Range HMBC Correlations to
1 to have AssembleIt consider up to this number of longer-range correlations below the
Weak threshold. Start the workwindow and in about 8 minutes (on a 3.06 GHz PC) AssembleIt reports 6 structures consistent with the specified strychnine data. They are saved in the file
Without the AssembleIt exhaustive listing of all possible structures, the first found structure might be regarded as the correct one. But display the
CC_corrs.plot.1 file of possible strychnine structures. All structures have the correct number of carbons and nitrogens, correct atom valences, and explain the observed HSQC and HMBC correlations within the specified limits. One of these structures is likely the correct carbon and nitrogen skeleton of strychnine. But further information is helpful to identify which one is the correct one.
The HMBC 2-bond (2JC,H), 3-bond (3JC,H), and longer-range (nJC,H) correlation ambiguity cannot be resolved experimentally. A valuable source of unambiguous correlation information is an additional DQF-COSY spectrum. HMBC can detect a bond if one of its bonded carbons is protonated. For DQF-COSY bond detection, both carbons need to be protonated. But then their vicinal coupling identifies their carbon-carbon bond.
gdqfcosy.fid/procparand run its transform. In the 2D Analysis workwindow, set
1, deselect the
[F1 Phase]switch for
-0.0017787* ppm, and
0.0905206* ppm. Run the 2D Analysis and it auto-runs the Report workwindow. In the Graphic workwindow, set the same phase functions as above, and display the
dqf-cosy.specfile and the correlation locations are also displayed as shown.
CC_corrs.plot. Reformat the circle diagram with the NMRgraph
[Redo Layout]and switch off the correlation labels through
[Hide Correlation Labels]to obtain the shown correlations display.
Return to spectrum type HMBC and the AssembleIt workwindow to add this DQF-COSY information. Set
2D: DQF-COSY to
DQF-COSY/report.log. Run this workwindow. Two possible strychnine structures result in a few seconds.
The first two tutorials cover the carbon-carbon bond detection from a 2D INADEQUATE spectrum. This would resolve remaining ambiguities. But it is over an order of magnitude less sensitive than HSQC, HMBC, and DQF-COSY spectra. The ADEQUATE acronym stands for Adequate sensitivity DoublE-QUAnTum spEctroscopy. The ADEQUATE experiment detects H-C-C moieties. It is known as 1,1-ADEQUATE due to the use of 1JH,C and 1JC,C couplings. Like 2D INADEQUATE, 1,1-ADEQUATE depends on both carbons involved being 13C. Using proton excitation and detection, ADEQUATE can be several times more sensitive than 2D INADEQUATE and this spectrum type can be acquired on an indirect detection probe.
Switch to the 1,1-ADEQUATE spectrum type. In the FFT workwindow load
adequate_dq.fid/procpar and adjust
F1: Start of Spectrum to
14597.577 (considering the +6.052 Hz referencing correction from the 1D carbon spectrum). Run this workwindow. In the 2D Analysis workwindow, set
Thresholds: Integral to
1200, to only consider stronger resonances. Run the workwindow and it auto-runs the Report workwindow. Load file
ADEQUATE/report.log in the Graphic and 2D Analysis workwindows to set the phase functions. Rerun the 2D Analysis workwindow and it auto-runs the Report workwindow.
adequate.specfile with correlation locations using the Graphic workwindow, as shown. Unaliased correlation areas are shown as black and F1 aliased correlations as green bounding boxes. Like 2D INADEQUATE, the 1,1-ADEQUATE signal-to-noise ratio is too limited to make spectral 1D projections useful. So the 1D proton spectrum is shown along the F2 axis. The 1D carbon spectrum does not have the double-quantum format of the F1 axis and is not shown.
CC_corrs.plot. Reformat the circle diagram with the NMRgraph
[Redo Layout]and switch off the correlation labels with
[Hide Correlation Labels]for the shown results.
ADEQUATE/report.logand run this workwindow. The structure determination takes under one second and two structures remain. Display the shown
CC_corrs.plot.1. Both structures are reported with four
Long-Range, and seven
The AssembleIt derived strychnine structure represents the possible carbon and nitrogen skeletons. From the edited HSQC, the resonance frequency and number of bonded hydrogen for each carbon are known, though they are not displayed. But it is known which carbons have remaining unexplained free valences. Free valences result from bonds to heteroatoms and higher bond multiplicities, which are not detected by the used NMR spectra. Based on the obtained molecular carbon chemical shift assignments, further predictions about bonded heteroatoms and higher bond orders can be derived from their expected effect on observed carbon chemical shifts.
To add heteroatoms to the previously displayed structures, select in NMRgraph
[Place Heteroatoms...]. In the started popup, confirm
O (oxygen) is selected as heteroatom to consider and click
[OK]. Oxygen atoms are placed in the structure to minimize the difference between observed and predicted carbon chemical shifts. The resulting structures are sorted by shift agreements.
C Shift Agreements...]. The average differences between the predicted and observed carbon shifts are
7.992ppm for the two structures. So the shown top structure is the most likely one and it agrees with the complete strychnine structure shown at the beginning of this tutorial.
Using only HMBC and HSQC derived ambiguous carbon-carbon correlations tends to become compute intensive for larger molecules where numerous candidate structures result. The chemical shift represents the chemical environment of each atom. So placing heteroatoms and sorting structures according to agreement of the carbon shifts with prediction can identify the most likely structure. In SECTION 6.4: "Strychnine Structures Derived From Previous Analysis Results", six possible structures result from HMBC and HSQC correlations. Placing heteroatoms and sorting these structures in carbon shift agreement order identifies the correct strychnine structure. In SECTION 6.5: "Using DQF-COSY", adding DQF-COSY information decreases the possible strychnine skeletons to two. Adding oxygen atoms and sorting the structures in carbon shift agreement order also identifies the correct strychnine skeleton. So 1,1-ADEQUATE information often is not needed for the structure elucidation of smaller molecules.
1Strychnine is an alkaloid extract obtained from the dried ripe seeds of Strychnos nux vomica, a small tree of the East Indies. In the past, strychnine has been used as an antiseptic, stomach tonic, circulatory stimulant, central nervous system stimulant, and medication for the relief of constipation. Strychnine is still in limited use today as a bird, mammal, and insect control agent.