AssembleIt
Combining Analysis Results from Multiple NMR Spectra
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The structure elucidation of minute quantities of organic material remains challenging. The most direct and unambiguous method for mapping the molecular carbon skeleton is through 2D INADEQUATE (Incredible Natural Abundance DoublE Quantum Transfer Experiment) spectra. Each identified spin system corresponds to a carbon-carbon bond and the display of all spin systems shows the identified carbon skeleton. The NMRanalyst page describes this automated spectrum analysis and carbon skeleton determination.

Heteronuclear 2D NMR spectra are an order of magnitude more sensitive to acquire than 2D INADEQUATE. But their interpretation is more challenging. The AssembleIt module described here is part of NMRanalyst. It is our solution for combining the information from different spectrum types and for deriving molecular skeletons from such ambiguous and often incomplete correlation information.

Deriving Carbon Skeletons from Heteronuclear NMR Spectra

The steps of this carbon skeleton determination are illustrated using the plant growth regulator gibberellic acid (MF: C19H22O6). The spectra used were acquired on a 500 MHz Varian instrument from a 7 mg/ml sample.

Step 1: Analysis of 1D Spectra

High resolution one-dimensional NMR spectra can be acquired with a modest amount of spectrometer time. The analysis of the 1D spectra provides valuable information for the structure analysis and helps to compensate for low resolution of the higher dimensional NMR spectra. NMRanalyst starts from the acquired FID and presents acquired information in numerical and graphical form. The following figure shows the overlaid experimental, NMRanalyst simulated, and the difference (residual) carbon spectra of gibberellic acid. The DMSO-d6 solvent resonances in the acquired spectrum are an order of magnitude higher than the gibberellic acid resonances. So NMRanalyst was used to subtract these solvent resonances from the spectrum and only their residuals around 40 ppm remain. The same analysis is done on the 1D proton spectrum.

1D Carbon Spectrum

Step 2: Analysis of HSQC Spectrum

Using the numerical 1D results, the multiplicity edited 2D HSQC (Heteronuclear Single Quantum Coherence) spectrum below is analyzed. It determines the direct carbon-proton bonds. The bounding boxes show the location of NMRanalyst identified correlations. The undetected resonance around 40 ppm in F1 and 2.5 ppm in F2 belongs to the solvent. The number of protons attached to each carbon is determined through the spectrum editing used. The CH2 groups appear negative. The distinction of the positive CH and CH3 groups is done based on the determined signal integral and the carbon and proton shifts.

HSQC Spectrum With Correlation Locations

Step 3: Analysis of HMBC Spectrum

A HMBC (Heteronuclear Multi-Bond Connectivity) NMR spectrum detects correlations between a proton and its surrounding carbon atoms. The strongest correlations are between nuclei two to three bonds apart. The bounding boxes indicate the location of identified correlations.

HMBC Spectrum With Correlation Locations

Step 4: Combining Spectral Analysis Results

The 1D carbon spectrum characterizes the distinguishable carbons. They are shown here sorted by their chemical shifts around the circle diagram. The edited HSQC results identify for each carbon the number and chemical shift of bonded protons. A red atom color implies no bonded protons (quaternary carbon), green one bonded proton, blue two bonded protons, and orange three bonded protons. The F1 frequency of an HMBC correlation identifies a carbon. Its F2 position is mapped by the corresponding HSQC correlation to its directly bonded carbon. This identifies a carbon-carbon correlation shown in the diagram by a dotted line. The line is dotted, because it could either correspond to a bond or a longer-range HMBC correlation. The arrow indicates the direction in which the correlation is detected. AssembleIt provides detailed information on how each correlation is derived from the spectral analysis results. AssembleIt can also use nitrogen NMR spectra and additional 1,1-ADEQUATE, DQF-COSY, and 2D INADEQUATE spectra.

HMBC Spectrum With Correlation Locations

Step 5: Deriving the Molecular Structure

Applying the AssembleIt module to the circle diagram above yields the shown gibberellic acid structure as the only one consistent with the specified correlations. The exhaustive structure generation takes only a few minutes on a modern PC. For other molecules numerous possible structures can result and AssembleIt reports them in decreasing likelihood. The six bonds labeled with a question mark were not observed experimentally. They were derived by AssembleIt based on detected long-range HMBC correlations. Sometimes 4-bond and longer-range HMBC correlations are observed. AssembleIt can be instructed to allow up to a user-specified number of such correlations in the structure elucidation. The displayed structure contains unexplained free carbon valences. Based on carbon chemical shift predication NMRanalyst can place unobserved heteroatoms at these positions (here keto- and hydroxyl-groups). For further information about AssembleIt, see the gibberellic acid and strychnine tutorials.


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