Introducing a Novel Class of Antibiotic
Darobactin A is an unusual strained heptapeptide with interesting biological properties. First isolated in 2019 from Photorhabdus khanii HGB1456, darobactin A possesses remarkable antibiotic activity against Gram-negative bacteria (including Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae).[1] Therefore, the compound is a promising platform for antibacterial drug development, particularly with the emergence of bacterial resistance to the existing frontline antibiotics.
A Novel Mode of Action
Gram-negative bacteria have an outer membrane that makes them resistant to many antibiotics. Darobactin A works by targeting BamA, which is an essential insertase protein in the formation of this outer membrane.[1,2] It is able to do this by mimicking a β-strand. It follows that the insertion and folding functions of the outer membrane proteins are disrupted, and thus bacteria become more penetrable.
The Synthetic Challenge
Photorhabdus khanii HGB1456 shows very limited production of darobactin A under laboratory cultivation conditions, and therefore chemical synthesis was required for further development of the compound. Chemists generally have no problem assembling amino acids into long peptide chains using solid-phase peptide synthesis (SPPS). In SPPS, Fmoc-protected building blocks are sequentially assembled onto a solid support, and excess building blocks and reagents can be conveniently filtered away at each step. In fact, this process is one of the few organic syntheses that can be fully automated.
Significantly, the total synthesis of the antibiotic Darobactin A cannot be achieved by standard SPPS, as heptapeptide assembly is complicated by the additional sidechain linkages of the tryptophan-like residues. These linkages result in strained macrocycles and the introduction of atrophisomerism into the molecule, which renders the molecule a considerable synthetic challenge.
Overcoming the Challenge Using the Larock Indole Synthesis
In the summer of 2022, two total syntheses of darobactin A were reported within one week of each other; the first by the group of Sarlah, the second by the group of Baran.[3,4] These independently-devised chemical syntheses utilised very similar synthetic strategies. Both papers showcased a succinct solution to the challenge of forming the two macrocycles, employing successive Larock reactions.[5] These reactions selectively join alkynes and aminohalo aryls to form indoles. In this case, they were used to form the strained macrocycles of darobactin A, while concurrently installing the required indole units of the tryptophan residues. Very similar building blocks were employed in both syntheses, as exemplified by the respective retrosyntheses of darobactin A shown in the figure below.
The key similarities and differences between the two syntheses are highlighted in the scheme below. Comparing the two syntheses, the Sarlah synthesis is shorter (16 steps vs 18 steps), while the Baran synthesis is higher yielding (26% vs <10%). The Sarlah synthesis showcases how the order of macrocycle formation is crucial for obtaining the correct atrophisomer, while the Baran synthesis allows late-stage modification of the amino acid residues on the eastern side of the molecule.
Summary
Darobactin A, a promising new class of antibiotics that targets the outer membrane of gram-negative bacteria, has been independently synthesised by two different research groups. This heptapeptide has demonstrated potent activity against a wide range of Gram-negative bacteria, including some that are resistant to currently available antibiotics. Furthermore, more active analogues of darobactin A have already been reported, some up to 32 times more potent than the parent compound (the reported syntheses can be readily adapted to vary the amino acid sequence).[6,7] The darobactin class of antibiotics could therefore represent a valuable addition to the current armoury available for the treatment of Gram-negative bacterial infections.
References
[1] A new antibiotic selectively kills Gram-negative pathogens. Imai Y, Meyer K.J., Iinishi A., Favre-Godal Q., Green R., Manuse S., Caboni M., Mori M., Niles S., Ghiglieri M., Honrao C., Ma X., Guo J.J., Makriyannis A., Linares-Otoya L., Böhringer N., Wuisan Z.G., Kaur H., Wu R., Mateus A., Typas A., Savitski M.M., Espinoza J.L., O'Rourke A., Nelson K.E., Hiller S., Noinaj N., Schäberle T.F., D'Onofrio A., Lewis K. Nature 2019, 576, 459. doi: 10.1038/s41586-019-1791-1.
[2] The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase. Kaur H., Jakob R.P., Marzinek J.K., Green R., Imai Y., Bolla J.R., Agustoni E., Robinson C.V., Bond P.J., Lewis K., Maier T., Hiller S. Nature. 2021, 593,125. doi: 10.1038/s41586-021-03455-w.
[3] Total Synthesis of Darobactin A. Nesic M., Ryffel D.B., Maturano J., Shevlin M., Pollack S.R., Gauthier D.R. Jr, Trigo-Mouriño P., Zhang L.K., Schultz D.M., McCabe Dunn J.M., Campeau L.C., Patel N.R., Petrone D.A., Sarlah D. J. Am. Chem. Soc. 2022,144, 14026. doi: 10.1021/jacs.2c05891.
[4] Atroposelective Total Synthesis of Darobactin A. Lin Y.C., Schneider F., Eberle K.J., Chiodi D., Nakamura H., Reisberg S.H., Chen J., Saito M., Baran P.S. J. Am. Chem. Soc. 2022, 17,14458. doi: 10.1021/jacs.2c05892
[5] Synthesis of 2,3-Disubstituted Indoles via Palladium-Catalyzed Annulation of Internal Alkynes. Larock R.C., Yum E.K., Re Fvik M.D., J. Org. Chem. 1998, 63, 7652, doi: 10.1021/jo9803277.
[6] Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering. Seyfert C.E., Porten C., Yuan B., Deckarm S., Panter F., Bader C.D., Coetzee J., Deschner F., Tehrani K.H.M.E., Higgins P.G., Seifert H., Marlovits T.C., Herrmann J., Müller R. Angew. Chem. Int. Ed. Engl. 2023, 62, e202214094. doi: 10.1002/anie.202214094.
[7] Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs. Böhringer N., Green R., Liu Y., Mettal U., Marner M., Modaresi S.M., Jakob R.P., Wuisan Z.G., Maier T., Iinishi A., Hiller S., Lewis K., Schäberle T.F. Microbiol. Spectr. 2021, 22, e0153521. doi: 10.1128/spectrum.01535-21.
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