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Current & Future Capabilities of NMR Spectroscopy in 3D Structure Elucidation

By Yalda Mobarhan, NMR Marketing & Communications Specialist, ACD/Labs

Following this year’s Small Molecule NMR Conference (SMASH), and hearing about the amount of attention paid to ACD/Structure Elucidator, I had an interesting conversation with Dimitris Argyropoulos, our NMR Business Manager. As an NMR spectroscopist I am aware of NMR’s unique capabilities when it comes to innovations in chemical and pharma industries, but for this blog post I was specifically interested in covering 3D structure elucidation and the technology involved in its current and future use.

Why is 3D structure elucidation using NMR becoming increasingly important?

A molecular structure drawn on a flat piece of paper may appear simple; however, molecules are not really lying flat. The exact structure of a molecule is determined by the type and connection of the atoms, their arrangement in space, and their relative distances. The same molecular formula can lead to different structural isomers—based on the bonding arrangements, or stereoisomers—depending on the number of stereocenters in the molecule. Despite sharing the same molecular formula, these isomers create huge discrepancies in chemical properties. 3D structure elucidation is both an essential and a challenging factor in R&D today due to the varying activities and impacts of the different stereoisomers; which goes beyond understanding the flat 2D molecular structure.

A tragic historic incident that highlights the importance of 3D structure elucidation is the well-known case of Thalidomide. Thalidomide was initially introduced and marketed in Germany in 1956 as an over the counter drug for treating morning sickness in pregnant women. In the years following its administration, about 10,000 infants worldwide were born with limb malformation or other defects since it was later found that the organic synthesis of Thalidomide had resulted in a racemic mixture of both (R)- and (S)-enantiomers interconverting quickly under biological conditions. In this particular case, the (R)-enantiomer has sedative effects, whereas the (S)-isomer is teratogenic. Although the compound is now approved as a cancer treatment, every measure is made to prevent previous incidents; like that from 1956. Thousands of people still suffer from the short period that the incorrect structure was administered to pregnant women. This disaster caused authorities in many countries to re-evaluate the control and approval of new pharmaceuticals.

Even with the development of Computer Assisted Structure Elucidation during the last 50 years, generally only 2D structures of a compound are resolved and the full 3D structure elucidation is more complicated. This task has traditionally been carried out using techniques like ECD or X-ray crystallography, while in the last 30 years NMR techniques like NOESY and ROESY have appeared that offer information about the spatial arrangement of atoms in a sample, providing an attractive alternative to previous techniques.

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Two ways scientists can analyze structure and function at or near atomic resolution are x-ray crystallography and NMR spectroscopy. What are the key differences between the two methods?

X-ray crystallography determines the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a 3D image of the density of electrons within the crystal is produced. This is an accurate method but has some difficulties experimentally, as scientists who want to pursue this method need to grow a crystal. This is often less than practical and does not reflect the natural status of the compound if present in cell, which is in solution.

NMR is a great alternative as it is the most powerful analytical tool for deducing the correct structure of newly isolated organic compounds. Also, with the help of modern NMR experiments such as NOESY and ROESY, one can evaluate the intensity of the NOE or ROE peaks in order to get a more quantitative idea about the actual distances between the involved nuclei. In addition, the molecules analyzed by NMR can be studied in a solution of a liquid crystal or some other ordered media with the freedom to move as they potentially would in their natural environment, but with the ability to extract the additional information because of the alignment imposed by the medium. Another major difference in these methods is the extent to which the data is analyzed. Its the interpretation of that data that’s tricky, which is where ACD/Labs’ NMR solutions come in to play.

 

Without technology, how did scientists previously interpret the data? How accurately can they interpret the data now?

Previously, we could only determine if the distance between atoms was more or less than 5 Angstroms. Now, using 1D or 2D NOESY and ROESY experiments, we are able to determine the exact distance between the atoms (<5 Angstroms), which is an important factor in understanding how the molecule appears in the 3D space.

With the advent of advanced computers and algorithms in recent years, this analysis has become possible and of increasing interest. In fact, ACD/Structure Elucidator v2019 introduces a new feature that allows scientists to precisely measure the distance between two atoms and characterize the 3D molecular structure. The software will calculate the distances of the atoms from the intensity of the NOESY or ROESY peaks and then also calculate all the possible conformers of the various possible stereoisomers. It will then try to match the experimentally determined distances to the calculated distances in the conformers. If there is a good match, then the conclusion can be drawn on which stereoisomer is the most probable. This can be easily done manually if the molecule has very few possible stereoisomers. If, however, there are many hundreds or thousands of possible stereoisomers then the manual analysis is very tedious, and here is where computers and advanced software come handy.

 

Where do you see the future of NMR research and software heading?

As science progresses, more regulations and requirements are being put in place to ensure drugs and products are safe for both humans and the environment. The amount of effort required to get a drug approved for the market today is more than that from 30 years ago. Chemical industry, including the drug and food industries, is under pressure to comply with regulatory requirements. Requirements will only increase and become stricter, and so will the daunting amount of data. It is characteristically said that if aspirin was discovered today it would have never gotten approval for human use.

On the other hand, in the fast paced and competitive industrial world today there is much more information available, all of which needs to be interpreted quickly, efficiently, and correctly to stay ahead of the game. Therefore, the ongoing effort will be to develop NMR software technology that allows faster and more efficient ways for data interpretation and decision-making. Software, like ACD/Structure Elucidator, can assist scientists with interpreting all this data and allows companies to adhere to the increasingly tighter regulations.

NMR plays a key role for determining the content and purity of a sample, as well as its molecular structure, both for research and industrial purposes. There are many more ways that the final 3D structure of a molecule can be elucidated than those discussed here, and we are trying to overcome many of the hurdles associated with this. Furthermore, it gets significantly more complicated as the size of the molecules increase and continues to be a work in progress. As science and software technologies continue to advance, we will learn even more about ACD/Labs’ NMR capabilities in the future.

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