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Chemical and ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded quinolines

Nature Chemistry volume 18, pages 61–72 (2026)Cite this article

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Abstract

Potent peptide ligands for therapeutically relevant targets are regularly returned from screening trillion-member libraries of ribosomally synthesized peptides containing non-canonical amino acids and macrocyclic architectures. Yet the chemical space explored by these peptides is a fraction of that embodied by natural products and pharmaceuticals, and most peptide leads require exhaustive medicinal chemistry optimization to improve potency and physicochemistry. To address the need for strategies to introduce chemical complexity and conformational control into peptide macrocycles, we report here that linear peptides with a reactive N-terminal β-keto or γ-keto amide can be synthesized ribosomally. Subsequent Friedländer reactions generate quinoline–peptide hybrids, some of which contain stable biaryl atropisomeric axes. We also demonstrate intramolecular Friedländer macrocyclization reactions—sufficiently mild to be employed on unprotected and in vitro-translated peptides—that embed a quinoline pharmacophore directly within the peptide backbone. The introduction of N-terminal ketone motifs into genetically encoded materials and their post-translational derivatization provides a paradigm for the programmed synthesis of peptide-derived materials that more closely resemble complex natural products.

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Fig. 1: Introduction of a reactive 1,3-dicarbonyl motif at the N terminus via IVT.
Fig. 2: Friedländer reactions of β-keto amides proceed readily in aqueous acetic acid.
Fig. 3: Post-translational Friedländer reactions on IVT products proceed under mild conditions.
Fig. 4: Friedländer reactions of β-keto amides generate stable atropisomers.
Fig. 5: Intramolecular post-translational Friedländer macrocyclization reactions.
Fig. 6: Aqueous Friedländer macrocyclization of N-methyl β-keto amide peptide LPa generates two distinct isomers.

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Data availability

FAIR data for this paper, including .fid files and other primary characterization data for all figures, are available via Figshare at https://doi.org/10.6084/m9.figshare.25968364 (ref. 112). Crystallographic data for the structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 2341552 ((M,S)-29), 2341553 ((P,S)-29) and 2395208 ((P)-CPa). Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk/structures. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Science Foundation (NSF) Center for Genetically Encoded Materials (C-GEM) grant CHE-2002182 (I.J.K., T.L.D., D.A.D., C.P., H.C., A.S. and S.J.M.) and the NSF Graduate Research Fellowship Program grants DGE-2139841 (T.L.D.) and DGE-1122492 (D.A.D.). C-GEM supported acylation and IVT experiments, as well as the synthesis, development and characterization of linear and macrocyclic peptides. MicroED experiments were supported by the National Institutes of Health (NIH) grant P41GM136508 (O.P., J.L. and T.G.) and funds from the Howard Hughes Medical Institute (O.P. and T.G.). This research made use of the Pines Magnetic Resonance Center’s Core NMR Facility (PMRC Core), as well as the Chemical and Biophysical Instrumentation Center (CBIC) at Yale University (RRID:SCR_021738), with equipment purchased with funds from NIH grant S10OD024998 (PMRC) or Yale University. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

We acknowledge H. Celik (PMRC Core), R. Giovine (PMRC Core) and R. E. Berry (CBIC) for assistance with NMR data acquisition; B. Q. Mercado (CBIC) and M. Houck (CBIC) for assistance with X-ray structural determination; and F. S. Menges (CBIC) for assistance with HRMS, MALDI-TOF and infrared data acquisition. We also acknowledge M. C. Guo (Yale) for additional assistance with infrared data acquisition, A. T. Champlin (Yale) for assistance with chiral semi-preparative HPLC, S. J. Hasnain (Yale) for preliminary contributions and members of the Schepartz and Miller groups for fruitful discussion.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Isaac J. Knudson, Cameron Paloutzian, Hannah Cho & Alanna Schepartz

  2. Department of Chemistry, Yale University, New Haven, CT, USA

    Taylor L. Dover, Diondra A. Dilworth & Scott J. Miller

  3. Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Orel Paz, Jieye Lin & Tamir Gonen

  4. Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Orel Paz & Tamir Gonen

  5. Department of Physiology, University of California, Los Angeles, Los Angeles, CA, USA

    Tamir Gonen

  6. Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA, USA

    Alanna Schepartz

  7. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA

    Alanna Schepartz

  8. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Alanna Schepartz

  9. Arc Institute, Palo Alto, CA, USA

    Alanna Schepartz

Authors
  1. Isaac J. Knudson
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  2. Taylor L. Dover
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  3. Diondra A. Dilworth
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  10. Scott J. Miller
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Contributions

I.J.K., D.A.D., A.S. and S.J.M. designed the project. I.J.K. led acylation experiments, Friedländer reaction optimization, IVT, SPPS and post-translational reactions. I.J.K., T.L.D. and D.A.D. synthesized materials and analysed/interpreted results. H.C. and C.P. assisted with acylation experiments. O.P., J.L. and T.G. collected and processed microED data. I.J.K., T.L.D., A.S. and S.J.M. prepared the paper.

Corresponding authors

Correspondence to Alanna Schepartz or Scott J. Miller.

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Competing interests

I.J.K., D.A.D., A.S. and S.J.M. have submitted a patent application, ‘Peptide Macrocycles with Embedded Heterocycles’ (2024, US patent application no. 63/559,854), related to the work disclosed here. The remaining authors declare no competing interests.

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Nature Chemistry thanks Julien Tailhades and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Flexizyme-promoted acylation of MH with DNB esters of fMet (fM) and monomers 1-4.

a-e, Abs260 chromatograms, extracted ion chromatograms, and deconvoluted mass spectra of the products resulting from flexizyme-promoted acylation of MH by the DNB esters of fMet (fM) and 1-4. In EIC, red traces correspond to the acylated MH, while black traces represent the unacylated MH. The red stars on the mass spectra indicate the acyl-MH peak in the deconvolution.

Source data

Extended Data Fig. 2 Peptides initiated with 2-tRNAfMet or 3-tRNAfMet were translated at levels comparable to those initiated with fMet-tRNAfMet.

Total ion chromatogram of IVT reactions programmed with a duplex DNA template encoding the FLAG-containing polypeptide MGVDYKDDDDK (MGV-flag) in the presence or absence of tRNAfMet acylated with the DNB esters of fMet (fM) or monomers 2 or 3 following anti-FLAG purification as described in the Supplementary Information, Sections III–IV. As previously described by others, we also observe side-products resulting from “drop-off-reinitiation”32,59,113,114,115,116.

Source data

Extended Data Fig. 3 Extracted ion chromatograms of Friedländer reactions of peptides synthesized using in vitro translation (IVT) methods.

a-d, Extracted mass chromatograms for 2-P1 (highlighted in grey) and each quinoline peptide (pink) after reaction in AcOH at 40 °C for 24 h. e, Extracted mass chromatograms for 2-P1 (grey) and presumed atropisomer quinoline peptide Q26-P1 (teal) after reaction in AcOH or in the presence of Yb(OTf)3 in EtOH or in MeCN. All reactions were run for 24 h at 40 °C.

Source data

Extended Data Fig. 4 Peptide stability and conversion in various Friedländer conditions.

a, Scheme of IVT reaction to produce peptides H2N-P1 and 2-P1, which were then exposed to either no reaction, AcOH at 40 °C for 24 h with no 2-aminoarylcarbonyl added, or AcOH at 40 °C for 24 h with 6 added. Extracted ion chromatogram (EIC) abundance for major ions of the starting material 2-P1 and product Q6-P1 (b), as well as the bystander peptide H2N-P1 (c). Bystander peptide H2N-P1 functions as an internal standard to demonstrate the stability of a nonreactive peptide under reaction conditions. AcOH reactions may result in mild degradation of 2-P1 but have little impact on H2N-P1. All reactions performed in triplicate, and all LC-HRMS measurements taken on the same day with the assumption that minimal changes in ionization efficiency occur during this timeframe. Error bars represent standard deviation (SD) d, Scheme showing reaction of IVT peptides with various 2-aminoarylcarbonyl substrates under three different Friedländer conditions. e, EIC abundance measurements show that Yb(OTf)3 reactions in organic solvents result in a more limited recovery of bystander peptide H2N-P1. EtOH conditions result in the complete disappearance of 2-P1, with a higher abundance of Q26-P1, while MeCN conditions show sizeable amounts of residual 2-P1 and a lower abundance of Q26-P1. All LC-HRMS measurements taken on the same day with the assumption that minimal changes in ionization efficiency occur across this timeframe. Note: experiments from a-c and d-e were performed separately with different batches of tRNA, IVT kit, monomer, and anti-FLAG magnetic beads and should not be directly compared.

Source data

Extended Data Fig. 5 The diastereomeric atropisomers of biaryl 29 show distinct chemical shifts and are chromatographically separable.

a, The crude 1H NMR spectrum in CDCl3 of biaryl 29 from Preparation II. The signals arising from the red protons in the accompanying structures were used to determine the diastereomeric ratio. b, The 1H NMR spectrum of purified biaryl 29 as a mixture of diastereomeric atropisomers in CDCl3. c, The 1H NMR spectrum of the minor diastereomeric atropisomer (M, S)-29 in CDCl3. d, The 1H NMR spectrum of the major diastereomeric atropisomer (P, S)-29 in CDCl3. The spectra shown are truncated for clarity. Additionally, the spectra have been manually aligned to the peaks at δ 2.70 and 2.68 ppm in panel (b) to account for minor deviations in chemical shift, presumably arising from differences in concentration and intermolecular interactions. Processed full spectra are provided within the Supplementary Information: “Processed NMR Spectra,” and raw .fid files are provided in the FAIR data.

Extended Data Fig. 6 Diagnostic shifts in the NMR spectra upon Friedländer macrocyclization.

a, Proton NMR of LP4 (red) and CP4 (teal) show the emergence of a distinct set of downfield aromatic protons (blue box) corresponding to the quinoline ring protons. b, gCOSY NMR spectra of LP4 (red, left) and CP4 (teal, right) display the correlated spin-couplings of the quinoline system. c, Carbon NMR of LP4 (red) and CP4 (teal) show the distinct loss of two ketone carbons around 200-210 ppm. Note: the tops of the solvent residuals have been truncated to save space; the truncation point is indicated with a dashed line.

Extended Data Fig. 7 Friedländer macrocyclization and DNA stability with high equivalents of Yb(OTf)3 in aqueous acetonitrile.

Scheme (a) and Total Ion Chromatograms (b-e) of Friedländer macrocyclization of LP2 (1 mM or 10 µM) with Yb(OTf)3 (1 mM, 10 mM, 100 mM or 200 mM) in H2O/MeCN (1:1 v/v). TIC conversions are averages of three independent replicates. Data is representative. Conversion of Friedländer macrocyclization is dramatically reduced in highly aqueous environments, but reactivity can be rescued with high equivalents of Yb(OTf)3. f, Stability of an oligonucleotide DNA:RNA duplex was assessed under aqueous acetonitrile Friedländer macrocyclization conditions in the presence of 200 mM Yb(OTf)3. Absorbance chromatogram (g) and extracted ion chromatogram (h) showing that RNA is degraded while DNA remains stable under reaction conditions. dFx was used as an internal standard to control for ionization efficiency across samples. Samples were run in triplicate, and representative traces are shown. i, Stability of RNA and DNA when exposed to reaction conditions. We suspect that the higher recovery of the DNA post-reaction is a result of improved DNA precipitation and column retention because of the Yb(OTf)3 salt compared to the unreacted sample. Error bars represent standard deviation (SD). j, Mass spectra of DNA before and after reaction are identical.

Source data

Extended Data Fig. 8 Structure and conversion between conformational isomers CPa1 and (P)-CPa.

a, Ramachandran plot of solid-state (P)-CPa. b, MicroED structure showing a double beta-turn motif (type II’ followed by type I). Cyclic backbone carbons highlighted in green. N-Methyl highlighted in orange. Intramolecular H-bonds indicated by orange dashed lines. H-bond distance is 2.30 Å between i + 3 Phe N-H and i carbonyl and 2.17 Å between i + 4 Kyn and i + 1 N-Me-Ala carbonyl. c, Scheme showing the thermal conversion of CPa1 to (P)-CPa. d, 1H NMR spectra show complete conversion of CPa1 to (P)-CPa after heating. The signal at δ 6.53 ppm in the bottom spectrum arises from the putative quinolinium proton.

Source data

Supplementary information

Supplementary Information

Supplementary Sections VI–VIII, Figs. 1–29, Tables 1–22, Methods, Discussion and processed NMR spectra.

Source data

Source Data Fig. 1

Tabulated A260 chromatograms, extracted ion chromatograms and mass spectra for Fig. 1c–h.

Source Data Fig. 2

Tabulated replicates of normalized A214 conversions.

Source Data Fig. 3

Tabulated extracted ion chromatograms and mass spectra for Fig. 3b–e.

Source Data Fig. 4

Tabulated extracted ion chromatograms and mass spectrum for Fig. 4c.

Source Data Fig. 5

Tabulated HPLC chromatograms for Fig. 5b–e and extracted ion chromatograms for Fig. 5g.

Source Data Fig. 6

Tabulated HPLC chromatogram for Fig. 6b and total ion chromatogram time points for Fig. 6c.

Source Data Extended Data Fig. 1

Tabulated A260 chromatograms, extracted ion chromatograms and mass spectra for Extended Data Fig. 1a–e.

Source Data Extended Data Fig. 2

Tabulated total ion chromatograms for Extended Data Fig. 2.

Source Data Extended Data Fig. 3

Tabulated extracted ion chromatograms for Extended Data Fig. 3a–e.

Source Data Extended Data Fig. 4

Tabulated extracted ion abundances for Extended Data Fig. 4b,c,e.

Source Data Extended Data Fig. 7

Tabulated total ion chromatograms for Extended Data Fig. 7b–e; and tabulated absorbance chromatograms, extracted ion chromatograms and mass spectra for Extended Data Fig. 7g–j.

Source Data Extended Data Fig. 8

Tabulated phi and psi angles for the Ramachandran plot in Extended Data Fig. 8a.

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Knudson, I.J., Dover, T.L., Dilworth, D.A. et al. Chemical and ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded quinolines. Nat. Chem. 18, 61–72 (2026). https://doi.org/10.1038/s41557-025-01935-4

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