XX TrxR Thioredoxin reductase
I. Introduction
5. Results and Discussion: Analysis and Identification of Compounds
5.1. The Truth of the Matter: Structure Elucidation through NMR Spectroscopy
“The world is full of obvious things that no one ever observes.”
Sherlock Holmes, Elementary In the laboratory, a chemist will base the outcome of a particular reaction or isolation on historical data, either from their own findings or on information gleaned from the literature. However, things do not always go according to plan, and, because of the capricious nature of chemistry, one cannot always assume that literature is correct. In some cases, novel synthetic or isolated compounds are identified, and in other cases, incorrect identification of compounds occurs. One such example of incorrect literature which has been corrected is found in aquatolide (Figure 5.1), a humulane-derived sesquiterpenoid lactone isolated from the daisy-like Asteriscus aquaticus.
O
H O H
O
H H
Figure 5.1. 2D structure of aquatolide and Asteriscus aquaticus.740
Initially characterized in 1989 by San Feliciano, et al.,741 it was thought, based on 1D and 2D NMR analysis, to contain a very rare [2]ladderane substructure (Figure 5.2). However, extensive experimental and theoretical quantum-chemical NMR analysis by Lodewyk, et al. in 2012742 showed that this novel compound did not contain the [2]ladderane core as previously reported, but rather it contained a bicyclo[2.1.1]hexane ring system, which was confirmed through X-ray crystallographic techniques. Other compounds which were initially incorrectly identified and have since been revised include asperjinone
135
and (+)-pestazaline B, and confusion still exists around the structure of the simpler 2,3,4,5-tetrahydro- 1,5-benzoxazepine-3-ol.743 In most cases this misidentification is through no conscious fault of the authors, rather it is due to ambiguous spectra and, as in the case of aquatolide, a lack of sensitivity in the equipment used to carry out the analyses. With the significant improvement in the technology behind these instruments, and the resultant increase in sensitivity, it is hoped that errors in the structural elucidation of compounds become less and less common.
O H
H
H O
H
O
Figure 5.2. Initial incorrect structure of aquatolide.
As characterization of large, novel molecules by NMR spectroscopy can be daunting, it is therefore convenient to analyze the building blocks which make up the large molecule separately, and then compare this data to the spectra obtained for the larger molecule. The correct identification of these precursors is vital in order to avoid the incorrect structural identification of the final products. Analysis of the final compounds synthesized previously made use of the NMR characterization of the synthesized compounds 62, 125, 126, 127 and the various chalcones, as well as spectra obtained for the commercially available starting materials. NMR spectral data for many of the compounds are available in the literature, however, there is a wide disparity in the field strengths at which the data have been acquired, in the solvents used, as well as the degree of completeness of the characterization. As such, each compound in this work has been completely characterized in DMSO-d6, with the use of D2O where solubility and stability dictated the use of a different solvent. This approach allowed for the identification of the peaks belonging to the moieties remaining in the final compounds based on their initial structures and the spectra thereof, and allows for the comparison of chemical shifts between different compounds, which may be used for assignment of spectral data of new compounds.704 This method can be demonstrated in the NMR analysis of compound 6, as it can be characterized based on data obtained from chalcone 71 and from 125 which in turn can be characterized based on information obtained from 62 (Scheme 5.1).
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Scheme 5.1. NMR structure elucidation of 6 through analysis of precursors.
O O
S O
O O-
Cl OH
O
Cl
O
O
S O
O O-
O O
6 71
125 62
The 1H NMR spectrum of 62 (Figure 5.3) allowed for the identification of the location of the methylene peak (Figure 5.3, peak b), as the relative integral ratios easily identify the signals corresponding to the protons of the methyl and methylene groups, both singlets with no 3J coupling. This NMR spectrum also establishes the peak pattern for the ortho-substituted acetophenone ring, both in fine structure and in the relative shifts. While literature spectra for compound 62 are available,744 the data is reported as acquired on a 200 MHz with the compound dissolved in CDCl3, and although the chemical shifts are reported, they have not been assigned to the structural fragments.
137
Figure 5.3. 1H NMR spectrum and assignment of 62.
As is often the case with this type of substitution, the fine structure arising from the ABCD spin system present in the ortho-substituted acetophenone ring is not resolved and the signals appear in a doublet- triplet-triplet-doublet pattern. While the order of these coupled partners is revealed by the COSY spectrum, the identity of the protons requires the use of a Nuclear Overhauser Enhancement (NOE) experiment (Figure 5.4) with selective irradiation of the methylene protons. This selective irradiation reveals through-space contacts to the acetophenone ring (Figure 5.4, peak b), from which the identity of the other protons of the acetophenone ring can be determined from the 3J COSY correlations. NOE interactions are also observed between the methylene protons and the methyl protons (Figure 5.4, peak a), as well as to the protons on the benzyl ring (Figure 5.4, peak c).
O
O a
b c
d
e f g
h
g
138
Figure 5.4. 1D NOE spectrum of 62 with selective irradiation of the methylene protons.
Comparison of the 13C and the DEPT135 spectra and analysis of the HMBC spectrum allowed for the identification and assignment of the quaternary carbons as well as the methyl, methylene and methine carbon atoms. HSQC analysis of this compound revealed that the most downfield proton in the 1H spectrum, which occupies the 6-position on the acetophenone ring, is not connected to the most downfield methine carbon atom in the 13C spectrum; rather the proton occupying the 4-position is connected to this carbon.
Following the characterization of 62, the analysis of the 1H spectrum of 125 (Figure 5.5) was relatively straightforward. The presence of the p-sulfonate group on the benzyl ring resulted in the simplification of the proton spectrum into the classic p-substituted phenyl ring pattern of two doublets (Figure 5.5, peaks e and h), corresponding to two protons each, rather than the poorly resolved multiplets seen for 62. Analysis of the NOESY spectrum (Figure 5.6) allowed for identification of the location of these two doublets, as a 3J COSY correlation exists between the methylene protons and the protons on the phenylsulfonate ring closer to the methylene, while no interaction is observed between the methylene protons and the protons closer to the sulfonate group.
O
O H
H H
H a b
c
139
Figure 5.5. 1H NMR spectrum and assignment of compound 125.
Figure 5.6. 1D NOE spectrum of 125 with selective irradiation of the methylene protons.
O
O
S O
O OK
H H
H a
b
O
O
S O
O OK
a
b c
d
e f
g
h
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As with 63, analysis and comparison of the 13C, DEPT135 and HMBC spectra obtained for 125 allowed for the identification and assignment of the carbon atoms, which showed very close correlation with the data obtained for 62, apart from the appearance of a new quaternary carbon and the subsequent loss of a methine carbon arising from the sulfonation of the benzyl ring. The data obtained from HSQC analysis of this compound corresponds with the data obtained for 62, with the most downfield carbon signal connected to the proton at position 4 on the acetophenone ring.
Analysis of 71 also made use of the information collected from the analyses of 62 and 125. As the protons present on the acetophenone ring had previously been identified, they could easily be distinguished in the 1H spectrum for 71 (Figure 5.7). Evidence for the presence of a trans double bond in the molecule was found in the existence of two one-proton doublets in the 1H spectrum (Figure 5.7, peaks d and f), with trans-bond characteristic J values of ca. 16 Hz.701 The NMR spectral data are reported in literature,699 however, as was the compound 62, no signal assignments have been made precluding a spectral comparison. As the benzaldehyde portion of the chalcone was derived from p- chlorobenzaldehyde, the proton signals for this ring appeared as two two-proton doublets, with the peaks differentiated through the presence (or lack) of NOESY correlations to the adjacent methine proton (Figure 5.8, peaks b and c). A COSY correlation, observed as a mixed-phase signal in the NOE spectrum (Figure 5.8, peak a), confirms the location of the signal corresponding to the two protons found on the benzaldehyde-derived phenyl ring.
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Figure 5.7. 1H NMR spectrum and assignment for chalcone 71.
Figure 5.8. 1D NOE spectrum of 71 with selective irradiation of the methine protons.
O
OH Cl
H H
H H
a
b c
O
OH Cl
a b
c
d f e g
a h
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Once the analyses of these compounds were complete, the analysis of the spectra obtained for 6 was relatively uncomplicated, as all of the peaks had previously been identified in at least one prior spectrum. The 1H NMR spectrum of 6 (Figure 5.9) shows the presence of a methylene peak (Figure 5.9, peak a) as well as the presence of the trans double bond protons (Figure 5.9, peaks d and f), indicating that both of these moieties are present in the molecule. The NOESY spectrum (Figure 5.10) again showed connections between the methylene proton and the acetophenone ring (highlighted in green), between the methylene protons and the sulfonate ring (red), as seen in the spectrum of 125, and between the methine proton of the double bond and the protons at the 2-position of the chlorophenyl ring (purple), as is seen in the spectrum of 69.
Figure 5.9. 1H NMR spectrum and assignment of 6.
O O
S O
O OK
Cl a
b
c d
e
f g
h i
j b
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Figure 5.10. 2D NOE spectrum of 6, showing connections between the various components.