Interview : Prof. Toshiaki Sunazuka "What are the molecular structures of "Splendid Gifts from Microorganisms"? Analysis is faster and easier now!

INTERVIEW 12

Director, Professor 　　　　　　　　
Omura Satoshi Memorial Institute
Graduate School of Infection Control Sciences, Kitasato University
Prof. Toshiaki Sunazuka

"What are the molecular structures of "Splendid Gifts from Microorganisms"? Analysis is faster and easier now!

Omura Satoshi Memorial Institute, Kitasato University has continued to discover new compounds every year, taking over the research theme of Dr. Satoshi Omura, a Nobel Prize winner in Physiology or Medicine in 2015. We interviewed Prof. Sunazuka, Director, about his research to find natural products that are produced by microorganisms for evaluation of their biological properties to discover drug lead compounds.

"The Omura Group is interested in unusual places where we could find new microorganisms: Isolated islands, the abyssal zone in the sea, and even places such as hot springs...".

Prof. Sunazuka, Director of Omura Satoshi Memorial Institute, Kitasato University explained the reason to search for unknown microorganisms. Some microorganisms produce secondary metabolites (bioactive substances) that serve as defensive to other living organisms. These compounds are essentially weapons for microorganisms to survive against their "enemies," for example other competitive microorganisms for their living positions. However, those secondary metabolites often have the medicinal potential that could also be beneficial for mankind.

Finding new microorganisms is a difficult task in itself, but to reach drug discovery, a number of additional stages are required. The initial stage is that the target microorganisms are isolated and cultured to evaluate biological properties of the produced secondary metabolites by the microorganisms. If the bioactivity of the culture broth is interesting and/or promising, the chemical structure of natural products in the broth will be analyzed. If the compound has potential to be a lead for medicinal chemistry, we embark chemical investigations through derivatization for a higher efficacy and lower toxicity, as well as total synthetic study to establish a versatile route to explore adjacent chemical space.

Each of these stages requires expertise with a high level of knowledge and skills. Currently, Omura Satoshi Memorial Institute consists of seven laboratories and four research centers (as of July 2023). When a promising candidate is identified, multiple groups in the institute work together to accelerate the research, like passing the baton in a relay. Such good teamwork has been proved to be very effective, and they succeed in continuously discovering new natural products at a rate of about 10 compounds per year.

Among the natural products in the Yellow Book, there are several whose molecular 3-D structures have not yet been elucidated.Basically, when we find an interesting compound, we want to know its molecular structure. This is because the information is required to elucidate the part causing bioactivity or to develop how to synthesize it.

To elucidate molecular structure, methods such as nuclear magnetic resonance spectrometry (NMR) and X-ray structure analysis used to be common, which was also the case with Omura Satoshi Memorial Institute, Kitasato University. In particular, using single crystal X-ray structure analysis makes it possible to clearly determine the 3D structure of a molecule from diffraction data of an X-ray. However, the bottleneck of this method is the need to prepare large (100um or more) single crystals without impurity. Natural products have complex and fluctuating structures, making it difficult to create large crystals. After much trial and error, it sometimes took six months to a year before data could be obtained.

In December 2021, electron diffractometer "XtaLAB Synergy-ED"(hereinafter, "Synergy-ED") was introduced by Omura Satoshi Memorial Institute, Kitasato University. The instrument employs a method called MicroED which is to irradiate electron beams, instead of X-rays, and use the diffraction data to elucidate the molecular structure. It is jointly developed by Rigaku Corporation who has X-ray diffraction technology and JEOL who has electron microscopy technology, and the system delivered to the institute is the very first machine.

The advantage of Synergy-ED is that it can perform structural analysis for micro crystal of several 10 nm to several 100 nm. Moreover, since the target area for measurement is so small, even if there are impurities, appropriate diffraction data can be obtained by avoiding them. The load and time to prepare a sample can be greatly reduced. It is expected to speed up the research process.

Several compounds have already been measured by using Synergy-ED at Omura Satoshi Memorial Institute, Kitasato University. Since it allows to elucidate molecular structure in a short time of period,"when we report a newly discovered compound in the paper, we can post the molecular structure in it and make the paper more convincing". (Prof. Sunazuka).

The introduction of Synergy-ED was triggered by a donation. A generous contribution was made by someone who was impressed by Dr. Omura winning the Nobel Prize. The amount of money was enough to afford an expensive instrument. While Director Sunazuka was pondering what to buy with the money and how to best use it for the benefit of everyone, Dr. Omura made one request.

"When I was a graduate student at the Tokyo University of Science, I had access to a 60 MHz high-performance nuclear magnetic resonance (NMR) spectrometer, the only one in Japan at the time. I acquired the know-how to operate and analyze this instrument, which gave me the confidence to determine the structure of compounds. This became a powerful weapon in my subsequent career as a researcher. Since we have received such a generous donation, why don't we purchase the most "groundbreaking" instrument available today?" It would be a stimulus for some researchers and a weapon for others. It would also be a memorial to Omura Satoshi Memorial Institute, Kitasato University. This is what he had in mind when he spoke.
Following this story, when Director Sunazuka was looking for a "groundbreaking" instrument, he heard that Synergy-ED was under development and decided to introduce it.

The MicroED technique used in Synergy-ED is a relatively new method, announced at the end of 2013 by Professor Tamir Gonen and his colleagues at the Howard Hughes Medical Institute. The ability to analyze the molecular structure of compounds for which only crystals as small as a few hundred nm can be obtained is truly groundbreaking. However, since it requires know-how of electron microscopy and crystal structure analysis, some people may feel it is a little bit difficult. Still, Synergy-ED is a ground-breaking instrument that has broadened the range of users by offering a seamless flow from measurement to analysis, making it a dedicated electron diffraction instrument that can be used even by non-specialists.

In fact, Kitasato University has had various people experience Synergy-ED. Some of them are undergraduate students. Not only researchers but also students are realizing that "anyone can elucidate molecular structures as long as they can make micro crystals," and this is becoming the norm. Synergy-ED may be one of the instruments that have created this new standard.

In the future, several analyses using Synergy-ED are projected at Omura Satoshi Memorial Institute.
They want to not only help use the results for analysis of new natural products and development of new research techniques, but also to elucidate all of the 3D structures for natural products in the "Yellow Book".
We want to decently understand the splendid gifts from microorganisms and accept them. By doing so, we may be able to understand the value of the gifts better.

Posted:November 2023

Electron Diffractometer XtaLAB Synergy-ED

Nuclear Magnetic Resonance Spectrometer (NMR)