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  • Review Article
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Synthesis and applications of mirror-image proteins

Nature Reviews Chemistry volume 7, pages 383–404 (2023)Cite this article

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Abstract

The homochirality of biomolecules in nature, such as DNA, RNA, peptides and proteins, has played a critical role in establishing and sustaining life on Earth. This chiral bias has also given synthetic chemists the opportunity to generate molecules with inverted chirality, unlocking valuable new properties and applications. Advances in the field of chemical protein synthesis have underpinned the generation of numerous ‘mirror-image’ proteins (those comprised entirely of d-amino acids instead of canonical l-amino acids), which cannot be accessed using recombinant expression technologies. This Review seeks to highlight recent work on synthetic mirror-image proteins, with a focus on modern synthetic strategies that have been leveraged to access these complex biomolecules as well as their applications in protein crystallography, drug discovery and the creation of mirror-image life.

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Fig. 1: Synthetic strategies for mirror-image proteins.
Fig. 2: The racemic and quasi-racemic protein X-ray crystallography workflow and application for accelerating crystallization of synthetic proteins.
Fig. 3: Mirror-image phage display: a powerful platform for generating d-peptide/protein binders against a target of interest.
Fig. 4: Additional mirror-image screening techniques.
Fig. 5: The mirror-image central dogma with synthetic schemes of key mirror-image d-polymerases, d-African swine fever virus polymerase X and mutant of Sulfolobus solfataricus d-Dpo4.
Fig. 6: Construction of critical componentry required for the generation of mirror-image life.
Fig. 7: Synthesis of mirror-image mutant split-protein T7 RNA polymerase and mirror-image DNA ligase.
Fig. 8: Approaches to creating a mirror-image artificial cell.

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References

  1. Pasteur, L. Mémoire sur la relation qui peut exister entre la forme cristalline et la composition chimique, et sur la cause de la polarisation rotatoire. C. R. Acad. Sci. 26, 535–538 (1848).

    Google Scholar 

  2. Snelders, H. A. M. in van’t Hoff-Le Bel Centennial Vol. 12, Ch. 5, 66–73 (American Chemical Society, 1975).

  3. Cahn, R. S. & Ingold, C. K. Specification of configuration about quadricovalent asymmetric atoms. J. Chem. Soc. 131, 612–622 (1951).

    Article  Google Scholar 

  4. Rosanoff, M. A. On Fischer’s classification of stereo-isomers. J. Am. Chem. Soc. 28, 114–121 (1906).

    Article  CAS  Google Scholar 

  5. Blackmond, D. G. The origin of biological homochirality. Cold Spring Harb. Perspect. Biol. 11, a032540 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xie, J. & Schultz, P. G. A chemical toolkit for proteins — an expanded genetic code. Nat. Rev. Mol. Cell Biol. 7, 775–782 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Rosano, G. L., Morales, E. S. & Ceccarelli, E. A. New tools for recombinant protein production in Escherichia coli: a 5‐year update. Protein Sci. 28, 1412–1422 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wright, T. H., Vallée, M. R. J. & Davis, B. G. From chemical mutagenesis to post‐expression mutagenesis: a 50 year odyssey. Angew. Chem. Int. Ed. 55, 5896–5903 (2016).

    Article  CAS  Google Scholar 

  9. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

    Article  CAS  PubMed  Google Scholar 

  10. Kreil, G. Conversion of l- to d-amino acids: a posttranslational reaction. Science 266, 996–997 (1994).

    Article  CAS  PubMed  Google Scholar 

  11. Mor, A., Amiche, M. & Nicolas, P. Enter a new post-translational modification: d-amino acids in gene-encoded peptides. Trends Biochem. Sci. 17, 481–485 (1992).

    Article  CAS  PubMed  Google Scholar 

  12. Liu, W. R., Wang, Y.-S. & Wan, W. Synthesis of proteins with defined posttranslational modifications using the genetic noncanonical amino acid incorporation approach. Mol. Biosyst. 7, 38–47 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Zhao, L. & Lu, W. Mirror image proteins. Curr. Opin. Chem. Biol. 22, 56–61 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Abboud, S. A., Cisse, E. H., Doudeau, M., Bénédetti, H. & Aucagne, V. A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation. Chem. Sci. 12, 3194–3201 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Giesler, R. J., Erickson, P. W. & Kay, M. S. Enhancing native chemical ligation for challenging chemical protein syntheses. Curr. Opin. Chem. Biol. 58, 37–44 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fuchs, O., Trunschke, S., Hanebrink, H., Reimann, M. & Seitz, O. Enabling cysteine‐free native chemical ligation at challenging junctions with a ligation auxiliary capable of base catalysis. Angew. Chem. Int. Ed. 60, 19483–19490 (2021).

    Article  CAS  Google Scholar 

  17. Mitchell, N. J. et al. Rapid additive-free selenocystine–selenoester peptide ligation. J. Am. Chem. Soc. 137, 14011–14014 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Zhang, Y., Xu, C., Lam, H. Y., Lee, C. L. & Li, X. Protein chemical synthesis by serine and threonine ligation. Proc. Natl Acad. Sci. USA 110, 6657–6662 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bang, D., Pentelute, B. L. & Kent, S. B. H. Kinetically controlled ligation for the convergent chemical synthesis of proteins. Angew. Chem. Int. Ed. 45, 3985–3988 (2006).

    Article  CAS  Google Scholar 

  20. Hackenberger, C. P. R. & Schwarzer, D. Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 47, 10030–10074 (2008).

    Article  CAS  Google Scholar 

  21. Saxon, E., Armstrong, J. I. & Bertozzi, C. R. A ‘traceless’ Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2, 2141–2143 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gao, X., Ma, Q. & Zhu, H. Distribution, industrial applications, and enzymatic synthesis of d-amino acids. Appl. Microbiol. Biotechnol. 99, 3341–3349 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, D.-P. et al. Highly selective synthesis of d-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory. Microb. Cell Fact. 20, 11 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Milton, R., Milton, S. & Kent, S. Total chemical synthesis of a d-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Science 256, 1445–1448 (1992). This publication marked the landmark synthesis of a mirror-image protein and provided critical experimental evidence demonstrating reciprocal chiral specificity of proteins.

    Article  CAS  PubMed  Google Scholar 

  26. Weinstock, M. T., Jacobsen, M. T. & Kay, M. S. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity. Proc. Natl Acad. Sci. USA 111, 11679–11684 (2014). This pivotal work pushed the frontier of chemical protein synthesis and uncovered the ambidextrous folding ability of the GroEL–ES chaperone, priming the field for development of a generalizable strategy for folding mirror-image proteins.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Petersen, M. E., Jacobsen, M. T. & Kay, M. S. Synthesis of tumor necrosis factor α for use as a mirror-image phage display target. Org. Biomol. Chem. 14, 5298–5303 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Feldmann, M. & Maini, R. N. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nat. Med. 9, 1245–1250 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Levinson, A. M. et al. Total chemical synthesis and folding of all-l and all-d variants of oncogenic KRas(G12V). J. Am. Chem. Soc. 139, 7632–7639 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wilson, R. M. et al. A fascinating journey into history: exploration of the world of isonitriles en route to complex amides. Angew. Chem. Int. Ed. 51, 2834–2848 (2012).

    Article  CAS  Google Scholar 

  31. Noguchi, T. et al. Synthesis of Grb2 SH2 domain proteins for mirror-image screening systems. Bioconjug. Chem. 28, 609–619 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Schmidt, N., Abendroth, F., Vázquez, O. & Hantschel, O. Synthesis of the l- and d-SH2 domain of the leukaemia oncogene Bcr-Abl. RSC Chem. Biol. 3, 1008–1012 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. De Rosa, L., Di Stasi, R. & D’Andrea, L. D. Total chemical synthesis by native chemical ligation of the all-d immunoglobulin-like domain 2 of Axl. Tetrahedron 75, 894–905 (2019).

    Article  Google Scholar 

  34. Callahan, A. J. et al. Single-shot flow synthesis of d-proteins for mirror-image phage display. ChemRxiv https://doi.org/10.26434/chemrxiv-2023-x86xp (2023).

    Article  Google Scholar 

  35. Kent, S. B. H. Novel protein science enabled by total chemical synthesis. Protein Sci. 28, 313–328 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. Yeung, H. et al. Radiation damage and racemic protein crystallography reveal the unique structure of the GASA/snakin protein superfamily. Angew. Chem. Int. Ed. 55, 7930–7933 (2016).

    Article  CAS  Google Scholar 

  37. Hung, L.-W., Kohmura, M., Ariyoshi, Y. & Kim, S.-H. Structural differences in d and l-monellin in the crystals of racemic mixture. J. Mol. Biol. 285, 311–321 (1999).

    Article  CAS  PubMed  Google Scholar 

  38. Payne, C. D. et al. Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides. RSC Chem. Biol. 2, 1682–1691 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Avital-Shmilovici, M. et al. Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. J. Am. Chem. Soc. 135, 3173–3185 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mandal, K., Pentelute, B. L., Tereshko, V., Kossiakoff, A. A. & Kent, S. B. H. X-ray structure of native scorpion toxin BmBKTx1 by racemic protein crystallography using direct methods. J. Am. Chem. Soc. 131, 1362–1363 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mandal, K. et al. Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods. Protein Sci. 18, 1146–1154 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pentelute, B. L. et al. Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography. Chem. Commun. 46, 8174–8176 (2010).

    Article  CAS  Google Scholar 

  43. Banigan, J. R. et al. Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography. Protein Sci. 19, 1840–1849 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Luisier, S., Avital-Shmilovici, M., Weiss, M. A. & Kent, S. B. H. Total chemical synthesis of human proinsulin. Chem. Commun. 46, 8177–8179 (2010).

    Article  CAS  Google Scholar 

  45. Dang, B. et al. Elucidation of the covalent and tertiary structures of biologically active Ts3 toxin. Angew. Chem. Int. Ed. 55, 8639–8642 (2016).

    Article  CAS  Google Scholar 

  46. Huang, Y. C. et al. Synthesis of l‐ and d‐ubiquitin by one‐pot ligation and metal‐free desulfurization. Chem. Eur. J. 22, 7623–7628 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Teng, P. et al. The folding propensity of α/sulfono-γ-AA peptidic foldamers with both left- and right-handedness. Commun. Chem. 4, 58 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Yeates, T. O. & Kent, S. B. H. Racemic protein crystallography. Annu. Rev. Biophys. 41, 41–61 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Mackay, A. L. Crystal enigma. Nature 342, 133 (1989).

    Article  Google Scholar 

  50. Zawadzke, L. E. & Berg, J. M. The structure of a centrosymmetric protein crystal. Proteins Struct. Funct. Genet. 16, 301–305 (1993).

    Article  CAS  PubMed  Google Scholar 

  51. Bunker, R. D. et al. A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography. Proc. Natl Acad. Sci. USA 112, 4310–4315 (2015). This example showcases the power of racemic protein X-ray crystallography for revealing important structural and functional information from targets that were previously intractable by standard techniques.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Voskuil, M. I. et al. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705–713 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Gupta, V. K., Kumar, M. M., Singh, D., Bisht, D. & Sharma, S. Drug targets in dormant Mycobacterium tuberculosis: can the conquest against tuberculosis become a reality? Infect. Dis. 50, 81–94 (2018).

    Article  Google Scholar 

  54. Chen, C.-C. et al. Racemic X-ray structure of l-type calcium channel antagonist Calciseptine prepared by total chemical synthesis. Sci. China Chem. 61, 702–707 (2018).

    Article  CAS  Google Scholar 

  55. Nandakumar, K. S. et al. A recombinant vaccine effectively induces C5a-specific neutralizing antibodies and prevents arthritis. PLoS ONE 5, e13511 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Zuo, C. et al. Chemical synthesis and racemic crystallization of rat C5a-desArg. Chin. Chem. Lett. 31, 693–696 (2020).

    Article  CAS  Google Scholar 

  57. Cook, W. J., Galakatos, N., Boyar, W. C., Walter, R. L. & Ealick, S. E. Structure of human desArg-C5a. Acta Crystallogr. D 66, 190–197 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Wang, C. K. et al. Mirror images of antimicrobial peptides provide reflections on their functions and amyloidogenic properties. J. Am. Chem. Soc. 138, 5706–5713 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. Kurgan, K. W. et al. Retention of native quaternary structure in racemic melittin crystals. J. Am. Chem. Soc. 141, 7704–7708 (2019). This article highlights the potential utility of racemic crystallography for the characterization of native polypeptide quaternary structures.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Anderson, D., Terwilliger, T. C., Wickner, W. & Eisenberg, D. Melittin forms crystals which are suitable for high resolution X-ray structural analysis and which reveal a molecular 2-fold axis of symmetry. J. Biol. Chem. 255, 2578–2582 (1980).

    Article  CAS  PubMed  Google Scholar 

  61. Yan, B., Ye, L., Xu, W. & Liu, L. Recent advances in racemic protein crystallography. Bioorg. Med. Chem. 25, 4953–4965 (2017).

    Article  CAS  PubMed  Google Scholar 

  62. Kent, S. B. Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis. Curr. Opin. Chem. Biol. 46, 1–9 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Qu, Q. et al. Synthesis of disulfide surrogate peptides incorporating large‐span surrogate bridges through a native‐chemical‐ligation‐assisted diaminodiacid strategy. Angew. Chem. Int. Ed. 59, 6037–6045 (2020).

    Article  CAS  Google Scholar 

  64. Pentelute, B. L., Gates, Z. P., Dashnau, J. L., Vanderkooi, J. M. & Kent, S. B. H. Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities. J. Am. Chem. Soc. 130, 9702–9707 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Pentelute, B. L. et al. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. J. Am. Chem. Soc. 130, 9695–9701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Okamoto, R. et al. (Quasi-)racemic X-ray structures of glycosylated and non-glycosylated forms of the chemokine Ser-CCL1 prepared by total chemical synthesis. Angew. Chem. Int. Ed. 53, 5194–5198 (2014).

    Article  CAS  Google Scholar 

  67. Dwek, R. A. Glycobiology: toward understanding the function of sugars. Chem. Rev. 96, 683–720 (1996).

    Article  CAS  PubMed  Google Scholar 

  68. Gao, S. et al. Monomer/oligomer quasi-racemic protein crystallography. J. Am. Chem. Soc. 138, 14497–14502 (2016). This work describes the application of quasi-racemic protein crystallography to facilitate the first successful crystallization of ubiquitin oligomers.

    Article  CAS  PubMed  Google Scholar 

  69. Beeton, C. et al. The d-diastereomer of ShK toxin selectively blocks voltage-gated K+ channels and inhibits T lymphocyte proliferation. J. Biol. Chem. 283, 988–997 (2008).

    Article  CAS  PubMed  Google Scholar 

  70. Dang, B., Kubota, T., Mandal, K., Bezanilla, F. & Kent, S. B. H. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography. J. Am. Chem. Soc. 135, 11911–11919 (2013).

    Article  CAS  PubMed  Google Scholar 

  71. Dang, B. et al. Inversion of the side-chain stereochemistry of indvidual Thr or Ile residues in a protein molecule: impact on the folding, stability, and structure of the ShK toxin. Angew. Chem. Int. Ed. 56, 3324–3328 (2017).

    Article  CAS  Google Scholar 

  72. Dang, B., Chhabra, S., Pennington, M. W., Norton, R. S. & Kent, S. B. H. Reinvestigation of the biological activity of d-allo-ShK protein. J. Biol. Chem. 292, 12599–12605 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Wulff, H., Castle, N. A. & Pardo, L. A. Voltage-gated potassium channels as therapeutic targets. Nat. Rev. Drug Discov. 8, 982–1001 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Kent, S. B. H. Inventing Synthetic Methods to Discover How Enzymes Work. 283–288 (GNT-Verlag GmbH, 2022).

  75. Li, Y., Cao, X. X., Tian, C. L. & Zheng, J. S. Chemical protein synthesis-assisted high-throughput screening strategies for D-peptides in drug discovery. Chin. Chem. Lett. 31, 2365–2374 (2020).

    Article  Google Scholar 

  76. D’Aloisio, V., Dognini, P., Hutcheon, G. A. & Coxon, C. R. PepTherDia: database and structural composition analysis of approved peptide therapeutics and diagnostics. Drug Discov. 26, 1409–1419 (2021).

    Google Scholar 

  77. Wang, L. et al. Therapeutic peptides: current applications and future directions. Sig. Transduct. Target. Ther. 7, 48 (2022).

    Article  CAS  Google Scholar 

  78. Lyu, X. et al. The global landscape of approved antibody therapies. Antib. Ther. 5, 233–257 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Muttenthaler, M., King, G. F., Adams, D. J. & Alewood, P. F. Trends in peptide drug discovery. Nat. Rev. Drug Discov. 20, 309–325 (2021).

    Article  CAS  PubMed  Google Scholar 

  80. Poduslo, J. F., Curran, G. L., Kumar, A., Frangione, B. & Soto, C. Beta-sheet breaker peptide inhibitor of Alzheimer’s amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma. J. Neurobiol. 39, 371–382 (1999).

    Article  CAS  PubMed  Google Scholar 

  81. Dintzis, H. M., Symer, D. E., Dintzis, R. Z., Zawadzke, L. E. & Berg, J. M. A comparison of the immunogenicity of a pair of enantiomeric proteins. Proteins 16, 306–308 (1993).

    Article  CAS  PubMed  Google Scholar 

  82. Darmostuk, M., Rimpelova, S., Gbelcova, H. & Ruml, T. Current approaches in SELEX: an update to aptamer selection technology. Biotechnol. Adv. 33, 1141–1161 (2015).

    Article  CAS  PubMed  Google Scholar 

  83. Scott, J. & Smith, G. Searching for peptide ligands with an epitope library. Science 249, 386–390 (1990).

    Article  CAS  PubMed  Google Scholar 

  84. Guo, Q.-Y. et al. Channel activity of mirror-image M2 proton channel of influenza A virus is blocked by achiral or chiral inhibitors. Protein Cell 10, 211–216 (2019).

    Article  CAS  PubMed  Google Scholar 

  85. Adaligil, E., Patil, K., Rodenstein, M. & Kumar, K. Discovery of peptide antibiotics composed of d-amino acids. ACS Chem. Biol. 14, 1498–1506 (2019).

    Article  CAS  PubMed  Google Scholar 

  86. Zhou, J., Du, X., Li, J., Yamagata, N. & Xu, B. Taurine boosts cellular uptake of small d-peptides for enzyme-instructed intracellular molecular self-assembly. J. Am. Chem. Soc. 137, 10040–10043 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Schumacher, T. N. et al. Identification of d-peptide ligands through mirror-image phage display. Science 271, 1854–1857 (1996). This seminal work established mirror-image phage display as a powerful technology for generating high-affinity d-configured peptide ligands of native l-proteins.

    Article  CAS  PubMed  Google Scholar 

  88. Wiesehan, K. et al. Selection of d-amino-acid peptides that bind to Alzheimer’s disease amyloid peptide Aβ142 by mirror image phage display. ChemBioChem 4, 748–753 (2003).

    Article  CAS  PubMed  Google Scholar 

  89. Fish, P. V., Steadman, D., Bayle, E. D. & Whiting, P. New approaches for the treatment of Alzheimer’s disease. Bioorg. Med. Chem. Lett. 29, 125–133 (2019).

    Article  CAS  PubMed  Google Scholar 

  90. Van Groen, T. et al. Reduction of Alzheimer’s disease amyloid plaque load in transgenic mice by D3, a d-enantiomeric peptide identified by mirror image phage display. ChemMedChem 3, 1848–1852 (2008).

    Article  PubMed  Google Scholar 

  91. Bocharov, E. V. et al. All-d-enantiomeric peptide D3 designed for Alzheimer’s disease treatment dynamically interacts with membrane-bound amyloid-β precursors. J. Med. Chem. 64, 16464–16479 (2021).

    Article  CAS  PubMed  Google Scholar 

  92. Kutzsche, J. et al. Safety and pharmacokinetics of the orally available antiprionic compound PRI-002: a single and multiple ascending dose phase I study. Alzheimers Dement. 6, e12001 (2020).

    Google Scholar 

  93. Funke, S. A. et al. Oral treatment with the d-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer’s disease transgenic mice. ACS Chem. Neurosci. 1, 639–648 (2010).

    Article  Google Scholar 

  94. Jiang, N. et al. Preclinical pharmacokinetic studies of the tritium labelled d-enantiomeric peptide D3 developed for the treatment of Alzheimer´s disease. PLoS ONE 10, e0128553 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  95. Leithold, L. H. E. et al. Pharmacokinetic properties of a novel d-peptide developed to be therapeutically active against toxic β-amyloid oligomers. Pharm. Res. 33, 328–336 (2016).

    Article  CAS  PubMed  Google Scholar 

  96. Van Groen, T. et al. The Aβ oligomer eliminating d-enantiomeric peptide RD2 improves cognition without changing plaque pathology. Sci. Rep. 7, 16275 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  97. Schemmert, S. et al. In vitro and in vivo efficacies of the linear and the cyclic version of an all-d-enantiomeric peptide developed for the treatment of Alzheimer’s disease. Int. J. Mol. Sci. 22, 6553 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Malhis, M. et al. Potent Tau aggregation inhibitor d-peptides selected against Tau-repeat 2 using mirror image phage display. ChemBioChem 22, 3049–3059 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Zhang, X. et al. Brain targeting and Aβ binding bifunctional nanoparticles inhibit amyloid protein aggregation in APP/PS1 transgenic mice. ACS Chem. Neurosci. 12, 2110–2121 (2021).

    Article  CAS  PubMed  Google Scholar 

  100. Brown, C. J., Lain, S., Verma, C. S., Fersht, A. R. & Lane, D. P. Awakening guardian angels: drugging the p53 pathway. Nat. Rev. Cancer 9, 862–873 (2009).

    Article  CAS  PubMed  Google Scholar 

  101. Pazgier, M. et al. Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX. Proc. Natl Acad. Sci. USA 106, 4665–4670 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Liu, M. et al. A left-handed solution to peptide inhibition of the p53–MDM2 interaction. Angew. Chem. Int. Ed. 49, 3649–3652 (2010).

    Article  CAS  Google Scholar 

  103. Liu, M. et al. d-peptide inhibitors of the p53–MDM2 interaction for targeted molecular therapy of malignant neoplasms. Proc. Natl Acad. Sci. USA 107, 14321–14326 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Chen, X. et al. A stapled peptide antagonist of MDM2 carried by polymeric micelles sensitizes glioblastoma to temozolomide treatment through p53 activation. J. Control. Rel. 218, 29–35 (2015).

    Article  CAS  Google Scholar 

  105. Bian, Z. et al. Awakening p53 in vivo by d-peptides-functionalized ultra-small nanoparticles: overcoming biological barriers to d-peptide drug delivery. Theranostics 8, 5320–5335 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Zhan, C. et al. An ultrahigh affinity d-peptide antagonist of MDM2. J. Med. Chem. 55, 6237–6241 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Kannan, S. et al. Macrocyclization of an all-d linear α-helical peptide imparts cellular permeability. Chem. Sci. 11, 5577–5591 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Noguchi, T. et al. Screening of a virtual mirror-image library of natural products. Chem. Commun. 52, 7653–7656 (2016).

    Article  CAS  Google Scholar 

  109. Shu, K. et al. Development of mirror-image screening systems for XIAP BIR3 domain inhibitors. Bioconjug. Chem. 30, 1395–1404 (2019).

    Article  CAS  PubMed  Google Scholar 

  110. Zhou, X. et al. A novel d‐peptide identified by mirror‐image phage display blocks TIGIT/PVR for cancer immunotherapy. Angew. Chem. Int. Ed. 59, 15114–15118 (2020).

    Article  CAS  Google Scholar 

  111. Chang, H. N. et al. Blocking of the PD‐1/PD‐L1 interaction by a d‐peptide antagonist for cancer immunotherapy. Angew. Chem. Int. Ed. 54, 11760–11764 (2015).

    Article  CAS  Google Scholar 

  112. Díaz‐Perlas, C. et al. Protein chemical synthesis combined with mirror‐image phage display yields d‐peptide EGF ligands that block the EGF–EGFR interaction. ChemBioChem 20, 2079–2084 (2019).

    Article  PubMed  Google Scholar 

  113. Eckert, D. M., Malashkevich, V. N., Hong, L. H., Carr, P. A. & Kim, P. S. Inhibiting HIV-1 entry. Cell 99, 103–115 (1999).

    Article  CAS  PubMed  Google Scholar 

  114. Welch, B. D., Vandemark, A. P., Heroux, A., Hill, C. P. & Kay, M. S. Potent d-peptide inhibitors of HIV-1 entry. Proc. Natl Acad. Sci. USA 104, 16828–16833 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Welch, B. D. et al. Design of a potent d-peptide HIV-1 entry inhibitor with a strong barrier to resistance. Virol. J. 84, 11235–11244 (2010).

    Article  CAS  Google Scholar 

  116. Redman, J. S. et al. Pharmacokinetic and chemical synthesis optimization of a potent d-peptide HIV entry inhibitor suitable for extended-release delivery. Mol. Pharm. 15, 1169–1179 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Mandal, K. et al. Chemical synthesis and X-ray structure of a heterochiral {d-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography. Proc. Natl Acad. Sci. USA 109, 14779–14784 (2012). This article expanded the scope of mirror-image phase display by demonstrating the value of screening protein (rather than peptide) libraries for generating ligands with excellent selectivity and affinity.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Uppalapati, M. et al. A potent d-protein antagonist of VEGF-A is nonimmunogenic, metabolically stable, and longer-circulating in vivo. ACS Chem. Biol. 11, 1058–1065 (2016).

    Article  CAS  PubMed  Google Scholar 

  119. Marinec, P. S. et al. A non-immunogenic bivalent d-protein potently inhibits retinal vascularization and tumor growth. ACS Chem. Biol. 16, 548–556 (2021).

    Article  CAS  PubMed  Google Scholar 

  120. Olea, C. Jr, Weidmann, J., Dawson, P. E. & Joyce, G. F. An l-RNA aptamer that binds and inhibits RNase. Chem. Biol. 22, 1437–1441 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Chen, J., Chen, M. & Zhu, T. F. Directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase. Nat. Biotechnol. 40, 1601–1609 (2022). This report discloses an innovative alternative strategy for the generation of mirror-image aptamer binders, relying on the panning of a mirror-image library against a native target of interest and amplification via mirror-image PCR.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Klußmann, S., Nolte, A., Bald, R., Erdmann, V. A. & Fürste, J. P. Mirror-image RNA that binds d-adenosine. Nat. Biotechnol. 14, 1112–1115 (1996). These back-to-back publications disclosed the development of selection-reflection as a next-generation combinatorial screening tool for the discovery of stable aptamer ligands.

    Article  PubMed  Google Scholar 

  123. Nolte, A., Klußmann, S., Bald, R., Erdmann, V. A. & Fürste, J. P. Mirror-design of l-oligonucleotide ligands binding to l-arginine. Nat. Biotechnol. 14, 1116–1119 (1996). These back-to-back publications disclosed the development of selection-reflection as a next-generation combinatorial screening tool for the discovery of stable aptamer ligands.

    Article  CAS  PubMed  Google Scholar 

  124. Williams, K. P. et al. Bioactive and nuclease-resistant l-DNA ligand of vasopressin. Proc. Natl Acad. Sci. USA 94, 11285–11290 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Purschke, W. G., Eulberg, D., Buchner, K., Vonhoff, S. & Klussmann, S. An l-RNA-based aquaretic agent that inhibits vasopressin in vivo. Proc. Natl Acad. Sci. USA 103, 5173–5178 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Sczepanski, J. T. & Joyce, G. F. Binding of a structured d-RNA molecule by an l-RNA aptamer. J. Am. Chem. Soc. 135, 13290–13293 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Umar, M. I. & Kwok, C. K. Specific suppression of d-RNA G-quadruplex–protein interaction with an l-RNA aptamer. Nucleic Acids Res. 48, 10125–10141 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Yatime, L. et al. Structural basis for the targeting of complement anaphylatoxin C5a using a mixed l-RNA/l-DNA aptamer. Nat. Commun. 6, 6481 (2015).

    Article  CAS  PubMed  Google Scholar 

  129. Rohden, F., Hoheisel, J. D. & Wieden, H.-J. Through the looking glass: milestones on the road towards mirroring life. Trends Biochem. Sci. 46, 931–943 (2021).

    Article  CAS  PubMed  Google Scholar 

  130. Wang, Z., Xu, W., Liu, L. & Zhu, T. F. A synthetic molecular system capable of mirror-image genetic replication and transcription. Nat. Chem. 8, 698–704 (2016).

    Article  CAS  PubMed  Google Scholar 

  131. Joyce, G. F. et al. Chiral selection in poly(C)-directed synthesis of oligo(G). Nature 310, 602–604 (1984).

    Article  CAS  PubMed  Google Scholar 

  132. Pech, A. et al. A thermostable d-polymerase for mirror-image PCR. Nucleic Acids Res. 45, 3997–4005 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Xu, W. et al. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction. Cell Discov. 3, 17008 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  134. Sherrer, S. M., Beyer, D. C., Xia, C. X., Fowler, J. D. & Suo, Z. Kinetic basis of sugar selection by a Y-family DNA polymerase from Sulfolobus solfataricus P2. Biochemistry 49, 10179–10186 (2010).

    Article  CAS  PubMed  Google Scholar 

  135. Jiang, W. et al. Mirror-image polymerase chain reaction. Cell Discov. 3, 17037 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Wang, M. et al. Mirror-image gene transcription and reverse transcription. Chem 5, 848–857 (2019).

    Article  CAS  Google Scholar 

  137. Fan, C., Deng, Q. & Zhu, T. F. Bioorthogonal information storage in l-DNA with a high-fidelity mirror-image Pfu DNA polymerase. Nat. Biotechnol. 39, 1548–1555 (2021).

    Article  CAS  PubMed  Google Scholar 

  138. Xiong, A. S. et al. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32, e98 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  139. Xu, Y. & Zhu, T. F. Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs. Science 378, 405–412 (2022). The assembly of a functional mirror-image T7 RNA polymerase represents a monumental feat of total chemical synthesis and a pivotal breakthrough on the journey towards mirror-image life.

    Article  CAS  PubMed  Google Scholar 

  140. Sczepanski, J. T. & Joyce, G. F. A cross-chiral RNA polymerase ribozyme. Nature 515, 440–442 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Tjhung, K. F., Sczepanski, J. T., Murtfeldt, E. R. & Joyce, G. F. RNA-catalyzed cross-chiral polymerization of RNA. J. Am. Chem. Soc. 142, 15331–15339 (2020).

    Article  CAS  PubMed  Google Scholar 

  142. Bare, G. A. L. & Joyce, G. F. Cross-chiral, RNA-catalyzed exponential amplification of RNA. J. Am. Chem. Soc. 143, 19160–19166 (2021).

    Article  CAS  PubMed  Google Scholar 

  143. Weidmann, J., Schnölzer, M., Dawson, P. E. & Hoheisel, J. D. Copying life: synthesis of an enzymatically active mirror-image DNA-ligase made of d-amino acids. Cell Chem. Biol. 26, 645–651 (2019).

    Article  CAS  PubMed  Google Scholar 

  144. Ling, J. J. et al. Mirror‐image 5S ribonucleoprotein complexes. Angew. Chem. Int. Ed. 59, 3724–3731 (2020).

    Article  CAS  Google Scholar 

  145. Jin, X., Park, O.-J. & Hong, S. H. Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications. Appl. Microbiol. Biotechnol. 103, 2947–2958 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Li, X. & Liu, C. C. Biological applications of expanded genetic codes. ChemBioChem 15, 2335–2341 (2014).

    Article  CAS  PubMed  Google Scholar 

  147. Katoh, T., Passioura, T. & Suga, H. Advances in in vitro genetic code reprogramming in 2014–2017. Synth. Biol. 3, ysy008 (2018).

    Article  CAS  Google Scholar 

  148. Katoh, T., Tajima, K. & Suga, H. Consecutive elongation of d-amino acids in translation. Cell Chem. Biol. 24, 46–54 (2017).

    Article  CAS  PubMed  Google Scholar 

  149. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).

    Article  CAS  PubMed  Google Scholar 

  150. Chen, J., Chen, M. & Zhu, T. F. Translating protein enzymes without aminoacyl-tRNA synthetases. Chem 7, 786–798 (2021).

    Article  CAS  Google Scholar 

  151. Murakami, H., Ohta, A., Ashigai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 3, 357–359 (2006).

    Article  CAS  PubMed  Google Scholar 

  152. Cameron, D. E., Bashor, C. J. & Collins, J. J. A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381–390 (2014).

    Article  CAS  PubMed  Google Scholar 

  153. Bedau, M. et al. Life after the synthetic cell. Nature 465, 422–424 (2010).

    Article  PubMed  Google Scholar 

  154. Fanalista, F. et al. Shape and size control of artificial cells for bottom-up biology. ACS Nano 13, 5439–5450 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    Article  CAS  PubMed  Google Scholar 

  156. van Nies, P. et al. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nat. Commun. 9, 1583 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  157. Murase, Y., Nakanishi, H., Tsuji, G., Sunami, T. & Ichihashi, N. In vitro evolution of unmodified 16S rRNA for simple ribosome reconstitution. ACS Synth. Biol. 7, 576–583 (2018).

    Article  CAS  PubMed  Google Scholar 

  158. Mirwaldt, C., Korndorfer, I. & Huber, R. The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 Å resolution. J. Mol. Biol. 246, 227–239 (1995).

    Article  CAS  PubMed  Google Scholar 

  159. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).

    Article  CAS  Google Scholar 

  160. Conibear, A. C., Watson, E. E., Payne, R. J. & Becker, C. F. W. Native chemical ligation in protein synthesis and semi-synthesis. Chem. Soc. Rev. 47, 9046–9068 (2018).

    Article  CAS  PubMed  Google Scholar 

  161. Kulkarni, S. S., Sayers, J., Premdjee, B. & Payne, R. J. Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat. Rev. Chem. 2, 0122 (2018).

    Article  CAS  Google Scholar 

  162. Bondalapati, S., Jbara, M. & Brik, A. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nat. Chem. 8, 407–418 (2016).

    Article  CAS  PubMed  Google Scholar 

  163. Thompson, R. E. & Muir, T. W. Chemoenzymatic semisynthesis of proteins. Chem. Rev. 120, 3051–3126 (2020).

    Article  CAS  PubMed  Google Scholar 

  164. Agouridas, V. et al. Native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations. Chem. Rev. 119, 7328–7443 (2019).

    Article  CAS  PubMed  Google Scholar 

  165. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994). The trailblazing development of the native chemical ligation technology underpins the area of chemical protein synthesis and has enabled construction of larger and more complex polypeptide architectures.

    Article  CAS  PubMed  Google Scholar 

  166. Wan, Q. & Danishefsky, S. J. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 46, 9248–9252 (2007).

    Article  CAS  Google Scholar 

  167. Jin, K., Li, T., Chow, H. Y., Liu, H. & Li, X. P−B desulfurization: an enabling method for protein chemical synthesis and site-specific deuteration. Angew. Chem. Int. Ed. 56, 14607–14611 (2017).

    Article  CAS  Google Scholar 

  168. Chisholm, T. S., Clayton, D., Dowman, L. J., Sayers, J. & Payne, R. J. Native chemical ligation–photodesulfurization in flow. J. Am. Chem. Soc. 140, 9020–9024 (2018).

    Article  CAS  PubMed  Google Scholar 

  169. Fang, G.-M. et al. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 50, 7645–7649 (2011).

    Article  CAS  Google Scholar 

  170. Zheng, J.-S., Tang, S., Qi, Y.-K., Wang, Z.-P. & Liu, L. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 8, 2483–2495 (2013).

    Article  CAS  PubMed  Google Scholar 

  171. Flood, D. T. et al. Leveraging the Knorr pyrazole synthesis for the facile generation of thioester surrogates for use in native chemical ligation. Angew. Chem. Int. Ed. 57, 11634–11639 (2018).

    Article  CAS  Google Scholar 

  172. Fang, G.-M., Wang, J.-X. & Liu, L. Convergent chemical synthesis of proteins by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 51, 10347–10350 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors acknowledge the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012) and the NHMRC Investigator Grant APP1174941 (to R.J.P.) for funding and the Research Training Scholarship Program for PhD support (to A.S.M., L.K. and J.W.C.M.).

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  1. School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia

    Katriona Harrison, Angus S. Mackay, Lucas Kambanis, Joshua W. C. Maxwell & Richard J. Payne

  2. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, New South Wales, Australia

    Katriona Harrison, Angus S. Mackay, Lucas Kambanis, Joshua W. C. Maxwell & Richard J. Payne

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Contributions

K.H. researched data for the article and contributed to writing, preparation of figures and reviewing and editing the manuscript. A.S.M. contributed to writing, preparation of figures and reviewing and editing the manuscript. L.K. and J.W.C.M contributed to preparation of the figures and revised the manuscript. R.J.P. revised the manuscript and conceived the overall direction of the manuscript. All authors have given approval to the final version of the manuscript.

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Harrison, K., Mackay, A.S., Kambanis, L. et al. Synthesis and applications of mirror-image proteins. Nat Rev Chem 7, 383–404 (2023). https://doi.org/10.1038/s41570-023-00493-y

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