By means of ligation chemistry—the “picking and mixing” of carefully designed precursors—it is possible to build large libraries of nonnatural but still bioactive peptide-like molecules. Although this approach eschews the usual biotech tools—organisms, enzymes, and reagents—it emulates the way nature makes new medicines. Specifically, it uses just a handful of building blocks to generate a great many compounds. The “fermentation” products, which are produced in aqueous media, may be screened directly for biological activity without any purification.

These assertions were made on behalf of a KAHA (α-ketoacid-hydroxylamine) ligation system developed by researchers at ETH-Zurich and the Institute of Transformative Bio-Molecules (ITbM) at Nagoya University. KAHA ligation enables highly efficient coupling between an α-ketoacid molecule and a hydroxylamine molecule by forming an amide (N(H)C=O) bond.

According to a press release issued by ITbM, the ligation system relies on an α-ketoacid initiator molecule, which is mixed with isooxazolidine elongation monomers. As monomers are added, a peptide chain grows. The peptide’s sequence depends on the choice of monomers, and the peptide’s length depends on the addition—at the end of the ligation procedure—of a terminator molecule.

Additional details on the procedure appeared September 7 Nature Chemistry, in an article entitled “Synthetic fermentation of bioactive nonribosomal peptides without organisms, enzymes, or reagents.” The article describes how researchers, led by Professor Jeffrey Bode, initially chose to work with 23 building blocks, including six initiators, eight monomers, and nine terminators. As a practical matter, these building blocks, in different combinations, are capable of producing around 30,000 compounds. The study ended up producing around 6,000.

“By increasing the number of building blocks, we can dramatically expand the number of potential compounds that can be formed,” explained Prof. Bode. The resulting “culture,” containing peptide chains, cyclohexanone byproducts and small amounts of the terminator (T) molecule, which is added in slight excess, can be diluted with buffer and directly subjected to biological screening. “To the best of our knowledge, there is currently no synthetic method to prepare such large numbers of compounds from relatively few starting materials by simply mixing building blocks followed by direct biological assays,” Prof. Bode added.

“No specialized knowledge of organic chemistry or handling of toxic material is required to produce complex organic molecules,” the authors of the Nature Chemistry article noted. “The ‘fermentations’ can be conducted in arrays and screened for biological activity without isolation or workup.”

Prof. Bode’s group demonstrated the applicability of synthetic fermentation by preparing synthetic cultures and screening them to find biologically active lead molecules for the inhibition of hepatitis C virus protease, which is believed to play a key role in the replication of the hepatitis C virus. As this enzyme lacks a well-defined pocket in its active site, this is a challenging target for finding bioactive hit molecules.

Ultimately, the investigators identified and characterized a hepatitis C virus NS3/4A protease inhibitor with a half-maximum inhibitory concentration of 1.0 μM.

“The hardest part of this work was looking beyond the entrenched idea that we needed single, pure compounds to do the biological screening. It was only when we looked at the way Nature makes new medicines—by producing dozens of similar compounds together—that we realized we could use our chemistry to do something very similar,” explained Prof. Bode. “By taking this inspiration, we found that we could make thousands of compounds from a few building blocks in a few hours, rather than the months it would normally take.”








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