Spectroscopic Studies Confirm Creation of Metalloenzyme with Novel Catalytic Functions
By Amy LairdMedia Inquiries
- Associate Dean for Communications, MCS
Enzymes are nature’s catalysts. They control chemical reactions with exquisite precision and speed, something that chemists strive—but often fail—to do with catalysts they create in the lab. In new research, a team of chemists from Johns Hopkins University, Carnegie Mellon University, and the Universitat de Girona bridged the worlds of biology and synthetic chemistry to harness the power of a metal-containing enzyme to perform a chemical synthesis that is faster, more enantioselective and more sustainable than the current synthetic approach.
The research results, published in Science, show how a naturally-occurring, nonheme iron-containing enzyme can be reconfigured to catalyze an azidation reaction. Azidation reactions are widely used in organic synthesis to create organoazides, a class of compounds found in a variety of biologically active pharmaceuticals.
“For nonheme enzymes, this is only the start,” said Associate Professor of Chemistry Yisong (Alex) Guo. “This study is one of the first to show that we can convert nonheme iron-containing enzymes to do this type of radical-initiated azide transfer chemistry.”
Azides (N3-) are used in a wide variety of organic reactions, including the development of many pharmaceutical compounds. But current synthetic approaches for azide installation are often hazardous, have low efficiency, and depend on toxic reagents and solvents. And, key to drug development, the current synthetic reaction cannot efficiently control which of the two possible enantiomers of the azidation product (a right-handed or left-handed configuration of the molecule) is created. Enantiomers have the same molecular formula, but their three-dimensional structures are mirror images of each other. Often only one configuration has the expected therapeutic effect, and, in some cases, the other may be harmful.
“Tailoring metalloenzymes—enzymes that contain metals—offers an advantage over the synthetic version of the catalyst because it gives us better leverage in controlling the reaction to produce a more enantioselective product,” Guo said. Enantioselectivity refers to the property of a chemical reaction in which the reaction product favors one enantiomer over the other.
Xiongyi Huang, an assistant professor of chemistry at Johns Hopkins University, started with a naturally occurring metalloenzyme that demonstrates radical relay C-H functionalization, which is similar to synthetic metal catalysts that carry out a non-natural C(sp3)‒H azidation reaction. He then used a process called directed evolution to speed up nature’s variation and selection process so that the original enzyme accrues beneficial mutations. The end result is a new and improved enzyme that has all of the beneficial features of the original but can now carry out the azidation reaction with high enantioselectivity — a reaction that doesn’t occur in nature.
To confirm that his bio-engineered enzyme followed similar reaction steps as its synthetic counterpart in the azidation reaction, Huang turned to the Guo lab, who are experts in using spectroscopy to understand the mechanisms of the chemical transformations catalyzed on the metal centers inside metalloproteins. Third year Ph.D. student Jared Paris led the effort to study the reaction progression using Mössbauer Spectroscopy, Electron Paramagnetic Resonance (EPR) Spectroscopy, and Ultraviolet–visible (UV–vis) spectroscopy. These studies provided key experimental evidence that the enzyme-mediated reaction proceeded similarly to the synthetic catalyst-mediated reaction.
“We needed to show evidence that the chemistry happening in the bio-engineered enzyme is not just some non-enzymatic event,” Guo explained. “This is where our spectroscopic data really played a key role in illustrating what actually happens. It was exactly what we expected to happen.”
Guo anticipates that using this and other bio-engineered metalloenzymes to do challenging catalysis will see many breakthroughs in the next decade.
“So far, the synthetic version is still very challenging to use and requires very toxic and harsh conditions. Hopefully new, bio-engineered enzymes can replace some of the synthetic catalysts to carve out a new direction in organic synthesis.”
Funding for the Carnegie Mellon portion of the study was provided by the National Science Foundation (CHE1654060).