Note pubs.acs.org/jnp
Structure Revision of Asperjinone Using Computer-Assisted Structure Elucidation Methods Mikhail Elyashberg,† Kirill Blinov,† Sergey Molodtsov,‡ and Antony J. Williams*,§ †
Advanced Chemistry Development, Moscow Department, 6 Akademik Bakulev Street, Moscow 117513, Russian Federation Novosibirsk Institute of Organic Chemistry, Siberian Division, Russian Academy of Sciences, 9 Akademik Lavrent’ev Avenue, Novosibirsk, 630090 Russian Federation § Royal Society of Chemistry, 904 Tamaras Circle, Wake Forest, North Carolina 27587, United States ‡
ABSTRACT: The elucidated structure of asperjinone (1), a natural product isolated from thermophilic Aspergillus terreus, was revised using the expert system Structure Elucidator. The reliability of the revised structure (2) was confirmed using 180 structures containing the (3,3-dimethyloxiran2-yl)methyl fragment (3) as a basis for comparison and whose chemical shifts contradict the suggested structure (1).
C
omputer-assisted structure elucidation (CASE)1,2 methods are widely used to identify the structures of newly isolated natural products as well as new products of organic synthesis. In the past decade it has been shown based on multiple comparisons2,3 that the most advanced CASE expert system is ACD/Structure Elucidator.3,4 The system was developed with the intention of elucidating the chemical structures of organic molecules from their MS and 1D and 2D NMR spectra, generally employed in combination. In the literature there are many examples documenting the successful application of Structure Elucidator, not only for the elucidation of complex natural products but also for the purpose of structure revision.5,6 Recently the successful computer-assisted structure elucidation of an organic synthesis product whose structure seemed undecipherable by traditional 2D NMR methods was described,7 while Codina et al.8 utilized the system for the analysis of a complex organic mixture. Further development of the system is driven primarily by continuously challenging the program with new structural problems described in the literature and, because of the general complexity of the compounds described, especially new compounds reported in the Journal of Natural Products. During the course of this work we utilized spectroscopic data reported by Liao et al.9 for deducing the structure of a new natural product named asperjinone 1 and presented in Figure 1. This compound was isolated, along with 12 other known compounds, from Aspergillus terreus. As a result of our analysis using Structure Elucidator, the structure of 1 was revised, and we suggest that structure 2 is the correct structure (see Figure 1). Even though an expert system in general mimics human thinking during the molecular structure elucidation process from spectroscopic data, the associated mathematical algorithms act in other ways. The program automatically forms a set © 2013 American Chemical Society and American Society of Pharmacognosy
Figure 1. Previously proposed structure of asperjinone (1) and the revised structure, 2.
of “axioms” and hypotheses on the basis of the available spectroscopic data and then deduces all (without any exception) structures that are logical corollaries of the initial set of “axioms”. The molecular formula C22H20O6 and the NMR data presented in Table 1 obtained from the reported work9 were used as input into the Structure Elucidator software. The molecular connectivity diagram (MCD) automatically created by the program is presented in Figure 2. The MCD shows atoms with their chemical shifts and their associated properties. These include the hybridization states and the possibility of neighboring with heteroatoms as well as HMBC connectivities between atoms. sp3-Hybridized carbons are colored in blue, sp2 are shown in violet, and atoms with Received: July 26, 2012 Published: January 4, 2013 113
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MCDC(119.0) and C(120.9)were classified as having ambiguous hybridization because the mentioned chemical shifts are characteristic both for the CC double bonds (sp2) and for the C(sp3) atom if it is included in an O−C−O fragment. Carbons with chemical shifts falling into the interval 152−167 ppm are likely connected in the molecule with at least one oxygen atom. The information presented in the MCD was used by the program for the purpose of structure generation.1 As a result, all structures in agreement with the HMBC correlations and atom properties were produced. No expert considerations common for a traditional approach regarding HMBC correlations were introduced. No structural inputs regarding the presence of aromatic rings or other conceivable rings in the structure were made. The following results from the structure generation process were obtained: k = 3658 → 2641 → 1939, tg = 1 min 50 s. This indicates that 3658 isomeric structures were generated in 1 min 50 s, and 2641 structures were stored on a disc after spectral and structural filtering.4 13C NMR chemical shifts were then calculated for the stored structures using an incremental approach10 (this procedure took 8 s), and duplicate structures were removed to give 1939 structures. During the latter procedure an isomer with the minimal deviation between the experimental and calculated chemical shifts was selected as the “best” representative of a set of identical structures. The output structural file was ranked in ascending order of the chemical shift deviation. 13C chemical shifts were predicted for all 1939 structures using a neural network-based program (14 s calculation time) and then for the first 15 structures of the ranked file using a HOSE code based program1 (1 min calculation time). The first nine structures of the ranked file are displayed in Figure 3. Atoms for which the Δ = |δCcalc − δCexpt| value, the difference between experimental and calculated chemical shifts, is less than 3 ppm are marked by green circles; yellow circles correspond to Δ = 3−15 ppm and red to Δ > 15 ppm. The figure shows that the first ranked structure (fully green) is characterized by the smallest deviations calculated by HOSE code and neural network based methods, while the structure proposed by Liao and co-workers9 was placed in third position by the ranking procedure. The deviation is almost twice the size of that given for the structure ranked in first position. To confirm the revised structure, 2, we performed a search for the (3,3-dimethyloxiran-2-yl)methyl fragment existing in structure 1 in the ACD/NMR database containing 425 000 structures with assigned 13C and 1H chemical shifts.
Table 1. 1D and 2D Spectroscopic Data Used for the Structure Elucidation of Asperjinone9 (600 MHz, acetoned6) δC
type
1 2 3 4 5
165.7 140.7 137.5 166.8 29.2
C C C C CH2
1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 7″
119.0 131.5 115.8 160.3 127.5 129.6 120.9 152.2 117.0 127.3 31.2
C CH CH C C CH C C CH CH CH2
68.8 77.0 19.7 25.3
CH C CH3 CH3
position
8″ 9″ 10″ 11″
δH (J in Hz)
HMBCa
3.97, d (11.2) 3.98, d (11.2)
C-2, 3, 4, 1 ″, 2″
7.63, d (8.1) 7.01, d (8.1)
C-2, 1 ′, 2′, 4′ C-1 ′, 4′
6.99, m
C-4″, 6″, 7″
6.66, 6.99, 2.67, 2.94, 3.76,
C-3″, 4″ C-5 C-2″, 3″, 4″, 8″, 9″
d (8.6) m dd (16.9, 8.0) dd (16.9, 5.0) m
1.22, s 1.33, s
C-8″, 9″, 11″ C-8″, 9″, 10″
a
HMBC correlations, optimized for 6 Hz, are from the proton(s) stated to the indicated carbon.
The program selected almost 180 structures, from which ca. 150 structures were chosen that exhibit the closest similarity with the environment of the oxirane fragment. For these structures, a scatter plot was created (see Figure 4). Here 13C chemical shifts related to the C-8′′ and C-9′′ atoms of structure 1 are presented for all selected structures. The chemical shift values (69 and 77 ppm) assigned to the corresponding atoms C-8′′ and C-9′′ in the original structure 1 are also shown by their labels on the right side of the graph.
Figure 2. The molecular connectivity diagram (MCD) extracted from the spectroscopic data. Atoms are artificially arranged in a manner that approximately corresponds to atom positions in revised structure 2.
ambiguous hybridization (sp3 or sp2) are colored in light blue. The symbol “ob” indicates that a given atom has a heteroatom as a neighbor. The symbol “fb” shows that such a heteroatom neighbor is forbidden. Two atoms (colored in pale blue) in the 114
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Figure 3. The first nine structures of the output file ranked by deviations calculated using neural network and HOSE code based 13C NMR prediction programs. Colored circles on the atoms display chemical shift differences. Green color denotes a difference of less than 3 ppm; yellow, between 3 and 15 ppm; and red, more than 15 ppm. Designation of deviations: dA, HOSE code based algorithm; dN, neural network based algorithm.
Inspection of the scatter plot convincingly confirms the incorrectness of the original structure: the chemical shifts of C8″ (68.8 ppm in structure 1) are observed in the range 60−65 ppm, while for C-9″ (77.0 ppm in structure 1) the corresponding range is 57−59 ppm. On the other hand, corroboration of the revised structure 2 was found in the Supporting Information of the original work.9 One of the compounds separated by the authors9 along with asperjinone (designated as butyrolactone V) was characterized, and its 13C and 1H NMR chemical shifts were assigned to the structure of butyrolactone V. This compound contains the revised structural component of structure 2. Both structures supplied with the assigned 13 C chemical shifts (for butyrolactone V only partial assignment is shown) are presented in Figure 5. The structure comparison leaves no doubts regarding the correctness of structure 2. Moreover, oxirane 1JCH couplings are typically ∼180 Hz, far larger than other oxygen-bearing
Figure 4. Scatter plot of the 13C chemical shift values related to atoms 8′′ and 9′′ of the original structure 1. Series 1 (blue circles) corresponds to atom 9′′ (δC 77 ppm in structure 1); series 2 (violet triangles), to atom 8′′ (δC 69 ppm in structure 1).
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(5) Williams, A. J.; Elyashberg, M. E.; Blinov, K. A.; Lankin, D. C.; Martin, G. E.; Reynolds, W. F.; Porco, J. A., Jr.; Singleton, C. A.; Su., S. J. Nat. Prod. 2008, 71, 581−588. (6) Elyashberg, M. E.; Williams, A. J.; Blinov, K. A. Nat. Prod. Rep. 2010, 27, 1296−1328. (7) Elyashberg, M. E.; Blinov, K. A.; Molodtsov, S. G.; Williams, A. J. Magn. Reson. Chem. 2012, 50, 22−27. (8) Codina, A.; Ryan, R. W.; Joyce, R.; Richards, D. S. Anal. Chem. 2010, 82, 9127−9133. (9) Liao, W.-Y.; Shen, C.-N.; Lin, L.-H.; Yang, Y.-L.; Han, H.-Y.; Chen, J.-W.; Kuo, S.-C.; Wu, S.-H.; Liaw, C.-C. J. Nat. Prod. 2012, 75, 630−635. (10) Smurnyy, Y. D.; Blinov, K. A.; Churanova, T. S.; Elyashberg, M. E.; Williams, A. J. J. Chem. Inf. Model. 2008, 48, 128−134.
Figure 5. Comparison of chemical shift in the revised part of structure 2 with those in butyrolactone V.
aliphatic carbons, and the existence of an oxirane ring in the asperjinone structure proved to be erroneous. We believe that the true structure of asperjinone is as shown in 2, that is, 3-[(3hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromen-6-yl)methyl]-4(4-hydroxyphenyl)furan-2,5-dione. The application of a CASE system to the structure elucidation of this natural product would have allowed the authors to avoid this incorrect structure as an output from their analysis. It should be noted that as far as we know this is the first example of a reliable structure revision being performed with the aid of only the CASE system, without additional experiments and quantum chemical NMR shift calculations. Our research shows how it is important to verify the structure of a new compound at least by NMR chemical shift prediction using fast and fully automatic empirical methods.1
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EXPERIMENTAL SECTION
All calculations were performed using the expert system ACD/ Structure Elucidator v.12 installed on a 2.8 GHz PC, with 3 Gb RAM.
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 (919) 201-1516. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Elyashberg, M. E.; Williams, A. J.; Blinov, K. A. Contemporary Computer-Assisted Approaches to Molecular Structure Elucidation; RSC Publishing: Cambridge, 2012. (2) Elyashberg, M. E.; Williams, A. J.; Martin, G. E. Prog. Nucl. Magn. Reson. Spectrosc. 2008, 53, 1−104. (3) Steinbeck, C. Nat. Prod. Rep. 2004, 21, 512−518. (4) Elyashberg, M. E.; Blinov, K. A.; Molodtsov, S. G.; Williams, A. J.; Martin, G. E. J. Chem. Inf. Comput. Sci. 2004, 44, 771−792. 116
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