Decoding Terukufazoline

Reflecting work in the Iwasaki Lab

Published here July 3, 2026

Discovery, Genome-Guided Structure Elucidation, and Total Synthesis of Terukufazoline A, a Macrocyclic Docosapeptide, from a Marine Cyanobacterium

Raimu Taguchi, Akira Ebihara, Yuta Tsunematsu, Ghulam Jeelani, Ryota Suzuki, Tomoyoshi Nozaki, Kiyotake Suenaga, and Arihiro Iwasaki

J. Am. Chem. Soc. 2026, XXXX, XXX–XXX. https://doi.org/10.1021/jacs.6c06625

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Large ribosomally synthesized and post-translationally modified peptides, RiPPs, push the practical limits of spectroscopic structure elucidation. Severe signal overlap in repetitive sequences, coexisting rotamers, and the absence of suitable crystals can defeat NMR, tandem MS, and diffraction-based methods in turn. Marine cyanobacteria are a particularly rich source of such structurally complex metabolites, yet many producers remain uncultured, making direct genomic access impossible without a metagenomics approach. The gap between biosynthetic potential and chemical characterization is widest for molecules large enough to exceed what conventional spectroscopy can resolve, a problem that calls for an integrated, multi-evidence strategy.

Researchers in the Iwasaki Lab at Chuo University and the Suenaga Lab at Keio University, published in J. Am. Chem. Soc., collected a marine cyanobacterium from Aguni Island, a remote Okinawan island, and isolated two novel macrocyclic peptides, terukufazolines A and B. Terukufazoline A proved immediately intractable: its 1H NMR spectrum in CD3OD showed severe signal overlap and a mixture of rotamers that persisted even after solvent screening. Partial acid hydrolysis, ozonolysis, derivatization, MicroED trials, and crystallization attempts all failed to establish the amino acid sequence. The team therefore combined spectroscopic analysis, shotgun metagenomic sequencing, chemical degradation, and convergent total synthesis to solve the structure.

Metagenomics recovered a patellamide-family biosynthetic gene cluster, tkf, most closely related to the tenuecyclamides cluster. The core peptide encoded by TkfE1, GFPPCAPCVPCGPCVPCGPCIC, matched exactly the 22 residues that NMR had identified in terukufazoline A: three Gly, Ala, two Val, Ile, Phe, one thiazole, six thiazolines, and seven Pro. With this sequence template in hand, reanalysis of the tandem MS data located the single thiazole unit adjacent to a Gly residue, completing the planar structure. Terukufazoline A is the first isolation-validated patellamide-type cyanobactin to reach the reported substrate-size ceiling of PatG-family macrocyclases, approximately 22 residues.

Absolute configuration determination required residue-by-residue interpretation of two complementary degradation methods. Ozonolysis followed by acid hydrolysis and chiral HPLC established L-configurations for all seven Pro residues and D-allo-Ile, but the thiazoline α-center proved prone to epimerization under these conditions. Direct acid hydrolysis in D2O/DCl with L-FDVDA derivatization showed no detectable deuterated Cys derivatives, confirming that the thiazoline α-centers carry the R configuration throughout. Total synthesis validated the complete stereostructure: a convergent fragment-coupling route used DEPBT and base-free allenone-mediated coupling to suppress epimerization at the labile thiazoline C-terminal carboxylic acid. Macrocyclization with DEPBT delivered synthetic terukufazoline A, whose NMR, IR, MS, and specific rotation data matched the natural product. Biological evaluation showed that both natural terukufazolines inhibit the growth of Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, with IC50 values of 4.1 μM and 11 μM respectively, while the acyclic cyclization precursors were appreciably less active, pointing to the macrocyclic scaffold as a contributor to antiparasitic potency.

The terukufazoline study provides a worked example of what becomes possible when metagenomics, degradation chemistry, and total synthesis operate in series rather than in parallel. The genome-guided sequencing step was not a shortcut; it was the essential bridge that made chemical validation tractable. For the broader natural products community, the work highlights a recurring pitfall in thiazoline-containing peptides: ozonolysis-hydrolysis can epimerize the thiazoline α-center, so direct acid hydrolysis should serve as the primary method when multiple thiazoline units are present. The convergent fragment-coupling strategy, with its explicit epimerization-suppression logic, also offers a transferable blueprint for synthesizing other azoline-rich cyanobactins that approach the macrocyclase size limit.

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Author

Akira Ebihara received his B.S. and M.S. degrees in chemistry from Keio University, Japan, in 2023, under the supervision of Professor Kiyotake Suenaga and Assistant Professor Arihiro Iwasaki. From his undergraduate through graduate studies, his research focused on natural products chemistry, particularly the isolation and structural elucidation of natural products with novel scaffolds and biological activities from marine cyanobacteria. He received the Excellent Poster Award at the 11th CSJ Chemistry Festa in 2021 and the Outstanding Achievement Award at the 68thToxin Symposium in 2022. He is currently a researcher in the Discovery Chemistry Department at Chugai Pharmaceutical Co., Ltd., Japan.

Author

Yuta Tsunematsu, Ph.D., obtained his doctoral degree in Pharmaceutical Sciences from Kyoto University under the supervision of Professor Hideaki Kakeya. His research focuses on natural product biosynthesis, genome mining, and synthetic biology-based drug discovery, with particular emphasis on the discovery and engineered production of bioactive natural products from microorganisms. After faculty appointments at the University of Shizuoka and a visiting scientist position at the Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, he joined Nagoya University in 2022. He is currently an Associate Professor at the Graduate School of Bioagricultural Sciences, Nagoya University.

Author

Kiyotake Suenaga, Ph.D., was born in Aizu-Wakamatsu, Fukushima, Japan, in 1969 and was raised in Sendai. After receiving his Ph.D. from Nagoya University in 1997 under Professor Kiyoyuki Yamada, he held academic positions at Nagoya University, the University of Shizuoka, and the University of Tsukuba before joining Keio University in 2006 as an associate professor. He was promoted to professor in 2015. He received the Chemical Society of Japan Award for Young Chemists in 2002. His research centers on marine natural products, with particular emphasis on the isolation, structural elucidation, total synthesis, and biological evaluation of bioactive secondary metabolites.

Author

Arihiro Iwasaki, Ph.D., earned his Ph.D. in Science from Keio University, Japan, where he trained in marine natural products chemistry under Professor Kiyotake Suenaga. Following appointments as Assistant Professor and Lecturer at Keio University, he joined Chuo University in 2023 to establish his independent research group. He was also a Visiting Researcher at the Scripps Institution of Oceanography, University of California San Diego. His research focuses on the discovery and structure elucidation of bioactive natural products from marine organisms. Dr. Iwasaki received the Young Scientists' Award from the Minister of Education, Culture, Sports, Science and Technology (MEXT) of Japan in 2024 for his contributions to marine natural products chemistry.

Decoding Terukufazoline

Author Spotlight

Author

Raimu Taguchi received his master’s degree from Keio University, where he studied natural products chemistry, including the isolation, structural elucidation, total synthesis, and biological evaluation of marine natural products, under the supervision of Professor Kiyotake Suenaga and then-Assistant Professor Arihiro Iwasaki. He is currently a Ph.D. student at the Graduate School of Bioagricultural Sciences, Nagoya University, under the supervision of Associate Professor Yuta Tsunematsu, and a JSPS Research Fellow (DC1). His current research focuses on genome-editing-based approaches to elucidate the biosynthetic origins of marine natural products.