Organic chemistry

Molecular structure assignment simplified

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An innovative combination of chemical synthesis, theory and spectroscopy could simplify determination of the structures of naturally occurring, biologically active molecules, which are often leads for drug discovery. See Letter p.436

Nature has long served as a source of biologically active molecules called natural products, many of which help to combat diseases1. In some instances, natural products have become approved drugs, whereas in others, close structural analogues have emerged as the optimal therapeutic agents. Knowledge of the molecular structure of a natural product is essential for drug-discovery efforts, but structure determination is still difficult for many complex molecules. On page 436, Wu et al.2 describe the re-elucidation of the molecular structures of the baulamycins A and B — two natural products that are potentially important leads for antibiotic discovery. The authors' approach could be applicable to determining the structures of other complex natural products.

When a drug is presented to a relevant biological receptor, the drug's unique chemical structure results in a selective binding event and a pharmacologically beneficial modulation of the receptor's function. Structural features that affect such binding include the sequence of chemical bonds in the drug and the array of chemical groups that define its molecular constitution. Perhaps the most essential structural feature is the 3D spatial (stereochemical) orientation of those bonds and groups. Molecules that contain one or more stereocentres, which are typically carbon atoms bonded to four different chemical appendages, can exist in multiple 3D forms called stereoisomers that often have distinct biological activities.

Baulamycin A and B were isolated from the bacterium Streptomyces tempisquensis in 2014, and their molecular structures were proposed3 (Fig. 1a). Each molecule contains 7 stereocentres and can therefore exist as one of 128 (27) stereoisomers. Wu et al. devised and implemented an efficient synthesis (comprising just ten steps) of the proposed structures, only to find that the spectroscopic properties of the resulting compounds were not identical to those of the natural materials. They concluded, as had researchers doing parallel work4, that the structures of the natural products had been misassigned — a not uncommon dilemma for chemists5. Wu and colleagues therefore undertook a series of studies that culminated in the deduction of the correct structure of the baulamycins (Fig. 1b).

Figure 1: Previously proposed and actual molecular structures of the baulamycins.

a, The molecular structures of baulamycin A and B were first proposed3 in 2014. Key features include the relative 3D geometrical alignments (stereochemical relationships) of the bonds, which are drawn as solid wedges (projecting above the plane of the page) or hashed wedges (projecting below). Pairs of bonds that project in the same direction are said to be in a syn relationship, whereas those that project in opposite directions are in an anti relationship. b, Wu et al.2 now report corrected structures for the baulamycins, which the authors confirmed by synthesizing the molecules. In the corrected structures, the carbon atoms (C), indicated by numbers, are called stereocentres. Variation of the relationships among these 7 stereocentres means that the baulamycins could take any of 128 different 3D forms (stereoisomers). The authors revised the relative geometry of the C14 and C1′ stereocentres on the basis of a parameter known as the coupling constant, or J value, which was obtained using nuclear magnetic resonance (NMR) spectroscopy. Deduction of the stereochemical relationship in the C11–C14 portion was guided by another NMR technique, called ROESY, and the stereochemical relationship in the C4–C8 region was determined by synthesizing a mixture of unequal amounts of stereoisomers and then comparing the NMR spectrum of the mixture with that of a natural sample of baulamycin A or B.

Nuclear magnetic resonance (NMR) spectroscopy is currently the most powerful spectroscopic method used to deduce the stereochemical features of organic compounds. A common approach for deducing the relative orientation of chemical groups in molecules is to capitalize on the dependence of an NMR parameter called the coupling constant (J) on the dihedral angle between two NMR-active atoms that interact through a small number of connecting bonds. However, molecules that lack rigidity exist as an ensemble of rapidly interconverting geometries (conformers). In such flexible structures, the J values are averaged over all contributing conformers, which makes the deduction of stereochemical relationships from J values less reliable than for rigid molecules. Indeed, the authors found that the J values alone were not sufficient to identify the correct structures of the conformationally flexible baulamycin A and B, which have 14 and 13 rotatable bonds, respectively, in their main carbon chains. This results in hundreds to thousands of relevant conformations, depending on the stereoisomer.

Another approach uses an NMR spectroscopy technique called ROESY, which exploits a phenomenon known as the nuclear Overhauser effect6 to identify hydrogen nuclei (specifically, protium isotopes, also known as protons) that are near enough to one another to interact 'through space' — meaning that the interaction does not occur through bonds. For example, ROESY can be used to identify groups that are on the same side of a molecule. This kind of analysis has conventionally been used for deducing structural relationships residing in a ring of atoms, rather than in acyclic regions of a molecule. Cyclic substructures have relatively few conformations in comparison to flexible molecules that contain chains of atoms, as present in the baulamycins.

Wu et al. performed an extensive computational study to identify the predominant conformations of four baulamycin stereoisomers that vary in the stereochemical relationships on their left-hand sides (specifically, the C14–C1′ and C11–C14 portions, using the numbering system in Fig. 1b). The team took advantage of a little-used, but potentially powerful, feature of ROESY for the study of conformationally unconstrained molecules7: they analysed ROESY data obtained previously3 from a natural sample of baulamycin A to deduce the interproton distances, which they then used to further refine the conformations that should be included in the calculations of a definitive set of J values. This allowed Wu and colleagues to propose the 3D arrangement of the atoms in the C11–C14 region of baulamycin A.

The authors also conceived an ingenious approach to determining the correct relative geometries of the remaining stereocentres in the baulamycins — C4, C6 and C8. The researchers synthesized a mixture of four stereoisomers of the baulamycins that have the reassigned structure in the C11–C14 region of the molecules. These molecules differed in their relative geometries from C4 to C8 and were made using the powerful assembly-line synthesis strategy8 reported previously by workers from the same laboratory. Through such an approach, stereoisomers can be produced in a predetermined but purposely unequal ratio, merely by controlling the stoichiometry of key reagents in the synthesis. From the carbon-13 NMR spectrum of that mixture, Wu et al. deduced the correct structure of baulamycin A by identifying the subset of signals that replicated those of the natural sample; on the basis of the intensities of the signals, which correlate with the amount of each stereoisomer in the mixture, the structure of the responsible stereoisomer was revealed immediately. By avoiding the need to individually synthesize all necessary stereoisomers, this approach greatly reduced the amount of work that was required. The authors confirmed that they had deduced the correct structure by synthesizing both baulamycin A and B, each as a single stereoisomer, and showing that each matched the NMR spectra of the natural samples.

The specific type of iterative synthesis used by Wu et al. is limited to the polypropionates (the class of natural product to which the baulamycins belong), but mixture synthesis9 in general warrants greater consideration as a strategy for determining the structures of complex molecules from other classes of natural product. The use of quantitative ROESY data in conjunction with computational studies also deserves greater attention for organic structure determination7. More broadly, the authors have demonstrated how success can be achieved by the clever and judicious integration of multiple existing techniques — a tactic always worthy of consideration in science.

Footnote 1


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Correspondence to Thomas R. Hoye.

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Thompson, S., Hoye, T. Molecular structure assignment simplified. Nature 547, 410–411 (2017) doi:10.1038/547410a

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