NMR based molecular structures from micrograms of natural compounds

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Author:

 

Christian Griesinger

Professor, Director and Scientific Member at MPI for Biophysical Chemistry, Germany

Contributing authors:

 

Nilamoni Nath

Assistant Professor, Gauhati University, India

Former PostDoc, MPI for Biophysical Chemistry, Germany

 

 Juan Carlos Fuentes-Monteverde

PostDoc, MPI for Biophysical Chemistry, Germany

Former Ph D student at Centro de Investigacións Científicas Avanzadas (CICA)

Universidade da Coruña, Spain

For many natural compounds, the molecular structure, i.e. constitution, configuration, and conformation, is often not known. Basic science and pharmaceutical research depend on the elucidation of the correct 3D structures of synthetic and natural compounds in the drug discovery process. While X-ray diffraction studies work well for molecules that crystallize, Nuclear Magnetic Resonance (NMR) spectroscopy remains the primary viable means of determining molecular structure. The three-dimensional structure can be obtained by deduction of dihedral angles and internuclear distances provided by the measurement of scalar J-couplings and Nuclear Overhauser effects (NOEs). However, these parameters may often fall short for the purpose when molecules are too flexible or stereogenic centers are separated by too many bonds.

Residual dipolar couplings (RDCs) and carbon residual chemical shift anisotropies (RCSAs, 1-3) offer structural restraints complementary to J-coupling and NOE, solving many challenging structures. However, for tiny sample amounts (< 400 micrograms), measurement of RDCs and RCSAs becomes insensitive due to the 1% abundance of 13C carbon. By contrast, owing to the high natural abundance of the 1H isotope, proton RCSAs can be measured from a few 10’s of micrograms  for natural compounds, and in combination with DFT calculations solve the three-dimensional structure.  

Figure 1: The glass tube on the left side of the figure contains a strychnine sample constrained in the colored PMMA gel. Spectrum in the right side of the figure is the 1H NMR spectrum of 10 microgram  strychnine in PMMA-d8 gel which is by and large free from polymer signals. Some of the resonances are enlarged as indicated by the arrows.

Proton RCSAs provide atom-specific information about the orientation of the proton’s bond with respect to the molecular coordinate system. We acquire them by soaking a solution of the molecule of interest in a chemically cross-linked polymer gel that is located in an NMR tube. By variation of the inner diameter of that tube, one condition with small RCSAs and one with large RCSAs is created, the difference of which provides RCSA difference values which are free from other contributions. A key feature in the present work was the synthesis of deuterated gels. Since they provide 1H spectra free of background signals, a few tenths of  micrograms of analyte can be measured.

Figure 2: Possible relative configurations of briarane B-3 which was isolated from Briareum asbestinum. The one with red colored R/S designations represents correct configuration (keeping the order C6, C7, C8, C9, C17, C1, C2, C10, C11).

For structure elucidation, RCSAs can be calculated for all possible structural candidates and then compared with the experimental values. The best-fitting structure then has the correct constitution, configuration, and conformation. If the structure of the molecule might be flexible and therefore a set of structures (i.e. a structural ensemble) might be required to explain the NMR data.

Our proton RCSA (Figure 3 and 4) based method, furnishes the correct configuration of several known natural products such as strychnine, brucine, santonin, and estrone as well as the flexible retrorsine compound. As a demanding application, proton RCSAs were utilized to obtain the configuration of 35 microgram of briarane B-3, a marine natural product already isolated in 1993 but still of unknown configuration.

Figure 3. Proton RCSA is the result of a collective effort of scientists. Here a part of the team on the occasion of the PhD thesis defense of JCFM. From left to right:  Prof. Dr. Michael Reggelin (Technische Universität Darmstadt, Germany), Christian Griesinger, Prof. Dr. Carlos Jiménez (Universidade da Coruña, Spain), Prof. Dr. Jaime Rodríguez (Universidade da Coruña, Spain), Juan Carlos Fuentes-Monteverde (JCFM), Dr. Dawrin Pech-Puch (postdoc at QUIMOLMAT research group, Spain)

Development of proton RCSA methodology along with the deuterated gels have lowered the required amount by approximately a factor 100 compared to approaches using RDCs or 13C RCSAs. The researchers expect that the method will have a big impact on natural product research.

Figure 4. From left to right: Professor Armando Navarro Vazquez and Juan Carlos Fuentes-Monteverde in pre-Coronatime after intense discussion about the manuscript at SMASH 2019 (Oporto, Portugal) and Nilamoni Nath, during a sample collection in the gardens of the Gauhati University (India).

We would like to share the link of a video about sample preparation here: https://youtu.be/z2THX9oVVwg and https://www.youtube.com/watch?v=C8cNWgJViGw  

To get more details, we invite you to read our article published in Nature Communications:

https://rdcu.be/b7yqC

References:

  1. Nath, N.; Schmidt, M.;Gil, R.; Williamson, R. T.; Martin, G. E.; Navarro-Vázquez, A.; Griesinger, C.; Liu, Y. J. Am. Chem. Soc. 138, 9548-9556 (2016).
  2. Hallwass, F.; Schmidt, M.; Sun, H.; Mazur, M.; Kummerlöwe, G.; Luy, Burkhard; Navarro-Vázquez, A.; Griesinger C.; Reinscheid, U. Chem. Int. Edit. 50, 9487-9490 (2011).
  3. Liu, Y; Navarro-Vázquez, A.; Gil, R.; Griesinger, C.; Martin, G. E.; Williamson, R. T. Nat. Protoc. 14, 217-247 (2019)

 

Christian Griesinger

Director, MPI for biophysical Chemistry

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