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The Story behind “Synergy of Synthesis, Computation, and NMR Reveals Correct Baulamycin Structures” Paula Lorenzo, Craig P. Butts,* Eddie L. Myers,*,† and Varinder K. Aggarwal* School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K.
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data from Sherman’s original investigation. We calculated the structures and NMR parameters for a series of fragment isomers of the baulamycins (including hundreds of conformations of each, resulting in several thousand calculations in total). Interestingly, we found that the calculations alone were not sufficiently accurate to solve the problem; in particular, they could not accurately predict the populations of the contributing conformations. However, by carefully refining the populations against some of the experimental NMR data (specifically nuclear Overhauser enhancement data), we obtained excellent computed models. Comparison of these models against the NMR data for the isolated natural product allowed us to unambiguously establish the relative configuration of the lefthand (“fragment A”, Figure 2) half of the molecule as structure 29 and thus eliminate 112 of the possible baulamycin structures. While a similar analysis of the right-hand half of the molecule strongly suggested that we could eliminate approximately half of the 16 remaining structures, limitations in the nuclear Overhauser enhancement data meant that we could not conduct the crucial population refinement and thus unequivocally determine the correct structure with NMR and computation alone. At this point, we leveraged the unique control that iterative homologation synthesis gives us in preparing the methyldecorated right-hand portion of the baulamycin structure with complete control over configuration at each step. This allowed us to prepare a predetermined mixture containing a 47:25:18:10 ratio of the four possible structures of the righthand half of the molecule. Comparison of the NMR data for this mixture against that of the reported natural product showed that the spectrum of the major 47% component of the mixture matched the spectrum of the baulamycins. The final step in the process was to determine the correct relationship between the two halves of the molecule, synthesize both mirror images of the resulting compound, and compare their optical rotations (a spectroscopic property that discriminates mirror image structures) to finally prepare the correct and unequivocal structure of the baulamycin molecules. There are a number of lessons that can be taken from this investigation. First, the structural complexity, combined with the conformational flexibility, makes this system a truly daunting challenge, but careful and quantitative consideration of NMR spectroscopic data provides much more detailed information about the chemical structure of natural products than even we had expected. Second, modern computational techniques allow us to leverage this detailed information encoded in the NMR spectra and rapidly eliminate a large
he ongoing battle against drug resistance is a major driver behind the search for new and diverse chemical structures from natural sources as potential leads into new medicines. One of the biggest challenges in this field is working out the molecular structure of these natural products, both their connectivity (which atoms are attached to which) and their configuration (how the atoms are arranged in three dimensions). If the molecules can be crystallized, then X-ray crystallography provides an unequivocal tool for determining these structures, but in the absence of this scientists are left with two primary weapons to elucidate chemical structure, chemical synthesis and NMR (nuclear magnetic resonance) spectroscopy. In a recent report,1 we have shown how careful and quantitative NMR analysis, combined with computation and chemical synthesis, can solve seemingly intractable problems in this realm. In 2014, David Sherman and his co-workers isolated two new natural products, baulamycins A and B, from the bacterium Streptomyces tempisquensis.2 These compounds were found to be active against the superbug methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus anthracis, through the inhibition of siderophore (iron chelator) biosynthesis, and their molecular structures were proposed solely on the basis of NMR spectroscopic measurements on the very small amounts of these compounds that could be isolated (Figure 1). This structure elucidation is incredibly challenging as even after the atomic connectivity is established, the baulamycins have a chain of 12 carbon atoms, 7 of which are stereogenic, giving 64 possible relative arrangements of these 7 substituents, and 2 “mirror image” (enantiomeric) forms of each arrangement, thus 128 possible chemical structures for the baulamycins. This is complicated further as the 12-carbon chain essentially behaves like a piece of soggy spaghetti in solution, interconverting between tens of thousands of different shapes (conformations) every few nanoseconds. Our contribution to this story started when we used our iterative homologation sequence4 to chemically synthesize the proposed structures of the baulamycins in a very efficient and flexible fashion, essentially growing the carbon chain one atom at a time with the substituents precisely oriented as we pleased. In late 2016, we completed this synthesis and found that while it had the correct connectivity, it appeared to have the wrong three-dimensional structure. This was confirmed and published in early 2017 by Guchhait et al.,3 and we are aware of other groups who found this problem in the ensuing months. At this stage, the critical question becomes “which of the 128 structures is the correct one?”. Making the remaining structures would require years of chemical synthesis, so instead we developed a plan to combine detailed computation of the stereoisomers with a quantitative re-examination of the NMR © XXXX American Chemical Society
Received: October 3, 2017
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DOI: 10.1021/acs.biochem.7b00994 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Proposed and revised structures of the baulamycins with a cartoon representation of a generalized siderophore mode of action.
Figure 2. Proposed structure of baulamycins 1 identifying the left-hand “fragment A” half of the molecule, with the tested structures 26−29. On the right is an overlay of hundreds of conformers of structure 29 for which NMR parameters were calculated and averaged before comparison against the reported experimental NMR data of the natural product. Reproduced from ref 1. Copyright 2017 Nature Publishing Group.
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majority of alternative chemical structures; without the calculations, this problem could not be even halfway solved in an efficient fashion. Finally, even with these powerful analytical and computational tools in hand (and in the absence of crystallography!), chemical synthesis provides the decisive means for determining the structure of natural products of this complexity.
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REFERENCES
(1) Wu, J., Lorenzo, P., Zhong, S., Ali, M., Butts, C. P., Myers, E. L., and Aggarwal, V. K. (2017) Synergy of synthesis, computation and NMR reveals correct baulamycin structures. Nature 547, 436−440. (2) Tripathi, A., Schofield, M. M., Chlipala, G. E., Schultz, P. J., Yim, I., Newmister, S. A., Nusca, T. D., Scaglione, J. B., Hanna, P. C., Tamayo-Castillo, G., and Sherman, D. H. (2014) Baulamycins A and B, broad-spectrum antibiotics identified as inhibitors of siderophore biosynthesis in Staphylococcus aureus and Bacillus anthracis. J. Am. Chem. Soc. 136, 1579−1586 [Erratum (2014) 136, 10541−10541]. (3) Guchhait, S., Chatterjee, S., Ampapathi, R. S., and Goswami, R. K. (2017) Total synthesis of reported structure of baulamycin A and its congeners. J. Org. Chem. 82, 2414−2435. (4) Burns, M., Essafi, S., Bame, J. R., Bull, S. P., Webster, M. P., Balieu, S., Dale, J. W., Butts, C. P., Harvey, J. N., and Aggarwal, V. K. (2014) Assembly-line synthesis of organic molecules with tailored shapes. Nature 513, 183−188.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Craig P. Butts: 0000-0001-6678-8839 Eddie L. Myers: 0000-0001-7742-4934 Varinder K. Aggarwal: 0000-0003-0344-6430 Present Address †
E.L.M.: School of Chemistry, NUI Galway, Galway, Ireland.
Author Contributions
All authors contributed equally. Notes
The authors declare no competing financial interest. B
DOI: 10.1021/acs.biochem.7b00994 Biochemistry XXXX, XXX, XXX−XXX
