Diselenide–selenoester ligation for chemical protein synthesis

Abstract

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide–selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method’s utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL–deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL–deselenization at β-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method’s speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master’s-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.

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Fig. 1: Overview of the DSL–deselenization protocol.
Fig. 2: Synthesis of adiponectin (19–107) conducted in the Payne Laboratory, The University of Sydney.
Fig. 3: Synthesis of haemathrin 1 conducted in the Payne Laboratory, the University of Sydney.
Fig. 4: Synthesis of haemathrin 2 conducted in the Payne Laboratory, the University of Sydney.
Fig. 5: Photographic representation of the DSL–deselenization procedure.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files

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Acknowledgements

We acknowledge funding from the Australian Research Council Linkage Scheme (LP150100308) to K.W.C.-F. and R.J.P.

Author information

Affiliations

Authors

Contributions

S.S.K. and E.E.W. performed the experiments, compound characterization and data analysis at the University of Sydney, Sydney Australia. B.P. replicated the selenoesterification and ligation experiments, compound characterization and data analysis at Novo Nordisk A/S, Denmark. S.S.K., E.E.W., B.P., K.W.C.-F. and R.J.P. contributed to experimental design and writing of the manuscript.

Corresponding author

Correspondence to Richard J. Payne.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Protocols thanks Michio Iwaoka and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Mitchell, N. J. et al. J. Am. Chem. Soc. 137, 14011–14014 (2015): https://doi.org/10.1021/jacs.5b07237

Mitchell, N. J. et al. Chem 2, 703–715 (2017): https://doi.org/10.1016/j.chempr.2017.04.003

Sayers, J. et al. J. Am. Chem. Soc. 140, 13327–13334 (2018): https://doi.org/10.1021/jacs.8b07877

Integrated supplementary information

Supplementary Fig. 1. Characterization of adiponectin (19–40) (C36Acm) selenoester—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin selenoester (19-40). Rt 4.67 min (0 to 50% B over 5 min, λ = 214 nm); Calculated Mass [2M+3H]3+: 1646.8, [M+2H]2+: 1235.3; Mass Found (ESI+): 1646.1 [2M+3H]3+, 1234.9[M+2H]2+.

Supplementary Fig. 2. Characterization of adiponectin 19–40-SePh (C36Acm)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin selenoester (19-40). Rt 2.93 min (5 to 30% B over 0.5 min followed by 30 to 50% B over 3.5 min, λ = 214 nm); Calculated Mass [M+2H]2+: 1235.1, [M+3H]3+: 823.7; Mass Found (ESI+): 1234.9 [M+2H]2+, 823.6 [M+3H]3+.

Supplementary Fig. 3. Characterization of adiponectin C-terminal diselenide dimer (41–107)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and B) ESI mass envelope of reverse-phase HPLC purified Adiponectin diselenide dimer (41–107); Rt 3.87 min (0 to 50% B over 5 min, λ = 214 nm). Calculated Mass [2M+7H]7+: 1875.5 [2M+8H]8+: 1641.2, [2M+9H]9+: 1459.0, [2M+10H]10+: 1313.2, [2M+11H]11+: 1193.9, [2M+12H]12+: 1094.4, [2M+13H]13+: 1010.4, [2M+14H]14+: 938.3, [2M+15H]15+: 875.8, [2M+16H]16+: 821.1, [2M+17H]17+: 772.9, [2M+18H]18+: 730.0, [2M+19H]19+: 691.6, [2M+20H]20+: 657.0 Mass Found (ESI+): 1874.9 [2M+7H]7+, 1640.6 [2M+8H]8+, 1458.2 [2M+9H]9+, 1312.8 [2M+10H]10+, 1193.6 [2M+11H]11+, 1094.3 [2M+12H]12+, 1010.2 [2M+13H]13+, 938.0 [2M+14H]14+, 875.5 [2M+15H]15+, 820.9 [2M+16H]16+, 772.6 [2M+17H]17+, 729.9 [2M+18H]18+, 691.4 [2M+19H]19+, 656.8 [2M+20H]20+.

Supplementary Fig. 4. DSL preparation of adiponectin (19–107) (C36Acm)—Payne Lab, University of Sydney.

Analytical reverse-phase HPLC trace of the crude Adiponectin (19–107) (C36Acm) a) post ligation and b) post deselenization.

Supplementary Fig. 5. Characterization of adiponectin (19–107) (C36Acm)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin (19–107) (C36Acm); Rt 4.22 min (0 to 50% B over 5 min, λ = 214 nm). Calculated Mass [M+5H]5+: 1759.1, [M+6H]6+: 1466.1, [M+7H]7+: 1256.8, [M+8H]8+: 1099.8, [M+9H]9+: 977.7, [M+10H]10+: 880.1, [M+11H]11+: 800.1; Mass Found (ESI+): 1758.7 [M+5H]5+, 1466.1 [M+6H]6+, 1256.8 [M+7H]7+, 1099.9 [M+8H]8+, 977.8 [M+9H]9+, 880.2 [M+10H]10+, 800.2 [M+11H]11+.

Supplementary Fig. 6. Acm deprotection of adiponectin (19–107) (C36Acm)—Payne Lab, University of Sydney.

Analytical reverse-phase HPLC trace of the crude Adiponectin (19–107) post-Acm deprotection.

Supplementary Fig. 7. Characterization of purified adiponectin (19–107)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin (19–107); Rt 4.48 min (0 to 50% B over 5 min, λ = 214 nm). Calculated Mass [M+5H]5+: 1745.3, [M+6H]6+: 1454.6, [M+7H]7+: 1246.9, [M+8H]8+: 1091.2, [M+9H]9+: 970.1, [M+10H]10+: 873.2, [M+11H]11+: 793.9, [M+12H]12+: 727.8; Mass Found (ESI+): 1745.1 [M+5H]5+, 1454.2 [M+6H]6+, 1246.8 [M+7H]7+, 1091.1 [M+8H]8+, 970.0 [M+9H]9+, 873.1[M+10H]10+, 793.8 [M+11H]11+, 727.8 [M+12H]12+.

Supplementary Fig. 8. MALDI-TOF of purified adiponectin (19–107)—Payne Lab, University of Sydney.

Calculated Mass for C372H588N114O125S2 [M+H]+: 8721.5; Mass Found: 8722.0 [M+H]+.

Supplementary Fig. 9. DSL preparation of adiponectin (19–107) (C36Acm)—Dep. Research Chemistry, Novo Nordisk.

Analytical reverse-phase HPLC (C18 BEH) of a) Crude DSL mixture during preparation of Adiponectin (19–107) (C36Acm) after 10 min b) Crude deselenization mixture of Adiponectin (19–107) (C36Acm) after 16 h (5% B over 0.5 min followed by 5 to 60% B over 3.5 min, λ = 214 nm).

Supplementary Fig. 10. Characterization of adiponectin (19–107) (C36Acm)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin (19–107) (C36Acm). Rt 2.48 min (5 to 20% B over 0.5 min followed by 20 to 40% B over 3.5 min, λ = 214 nm); Calculated Mass [M+5H]5+: 1759.5, [M+6H]6+: 1466.4, [M+7H]7+: 1257.1, [M+8H]8+: 1100.1, [M+9H]9+: 977.9, [M+10H]10+: 880.2, [M+11H]11+: 800.3, [M+12H]12+: 733.7; Mass Found (ESI+): 1759.3 [M+5H]5+, 1466.2 [M+6H]6+, 1256.9 [M+7H]7+, 1099.9 [M+8H]8+, 977.8 [M+9H]9+, 880.1 [M+10H]10+, 800.2 [M+11H]11+, 733.6 [M+12H]12+.

Supplementary Fig. 11. Acm deprotection of adiponectin (19–107) (C36Acm)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 CSH) and b) ESI mass envelope of reverse-phase HPLC purified Adiponectin (19–107). Rt 2.04 min (5% B over 0.5 min followed by 5 to 60% B over 3.5 min, λ = 214 nm); Calculated Mass [M+5H]5+: 1745.3, [M+6H]6+: 1454.6, [M+7H]7+: 1246.9, [M+8H]8+: 1091.2, [M+9H]9+: 970.0, [M+10H]10+: 873.1, [M+11H]11+: 793.9, [M+12H]12+: 727.8; Mass Found (ESI+): 1744.9 [M+5H]5+, 1454.4 [M+6H]6+, 1246.8 [M+7H]7+, 1091.1 [M+8H]8+, 970.0[M+9H]9+, 873.1 [M+10H]10+, 793.8 [M+11H]11+, 727.7 [M+12H]12+.

Supplementary Fig. 12. Characterization of haemathrin-1 N-terminal selenoester (1–23)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and B) ESI mass envelope of reverse-phase HPLC purified Haemathrin-1 selenoester (1–28). Rt 3.91 min (0 to 50% B over 5 min, λ = 214 nm); Calculated Mass [2M+3H]3+: 1856.9, [M+2H]2+: 1393.0, [M+3H]3+: 929.0, [M+4H]4+: 697.0, [M+5H]5+: 557.8, [M+6H]6+: 465.0; Mass Found (ESI+): 1856.2 [2M+3H]3+, 1392.2 [M+2H]2+, 928.5 [M+3H]3+, 696.7 [M+4H]4+, 557.5 [M+5H]5+, 464.7 [M+6H]6+.

Supplementary Fig. 13. Characterization of haemathrin-1 N-terminal selenoester (1–23)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-1 selenoester (1–23). Rt 2.21 min (5 to 20% B over 0.5 min followed by 20 to 40% B over 3.5 min, λ = 214 nm); Calculated Mass [M+2H]2+: 1392.7, [M+3H]3+: 928.8, [M+4H]4+: 696.8, [M+5H]5+: 557.7; Mass Found (ESI+): 1392.5 [M+2H]2+, 928.6 [M+3H]3+, 696.7 [M+4H]4+, 557.6 [M+5H]5+.

Supplementary Fig. 14. Characterization of haemathrin-1 C-terminal diselenide dimer (24–59)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-1 diselenide dimer (29–59); Rt 3.41 min (0 to 50% B over 5 min, λ = 215 nm). Calculated Mass [2M+5H]5+: 1658.3, [2M+6H]6+: 1382.1, [2M+7H]7+: 1184.8, [2M+8H]8+: 1036.8, [2M+9H]9+: 921.7, [2M+10H]10+: 829.6, [2M+11H]11+: 754.3, [2M+12H]12+: 691.5; Mass Found (ESI+); 1657.5 [2M+5H]5+, 1381.4 [2M+6H]6+, 1184.3 [2M+7H]7+, 1036.4 [2M+8H]8+, 921.3 [2M+9H]9+, 829.3 [2M+10H]10+, 754.0 [2M+11H]11+, 691.2 [2M+12H]12+.

Supplementary Fig. 15. Characterization of haemathrin-2 N-terminal selenoester (1–28)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and B) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2 selenoester (1–28). Rt 3.91 min (0 to 50% B over 5 min, λ = 214 nm); Calculated Mass [M+2H]2+: 1701.3, [M+3H]3+: 1134.5, [M+4H]4+: 851.2, [M+5H]5+: 681.1, [M+6H]6+: 567.8, [M+7H]7+: 486.8; Mass Found (ESI+): 1701.1 [M+2H]2+, 1134.4 [M+3H]3+, 851.0 [M+4H]4+, 681.1 [M+5H]5+, 567.7 [M+6H]6+, 486.8 [M+7H]7+.

Supplementary Fig. 16. Characterization of haemathrin-2 N-terminal selenoester (1–28)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2 selenoester (1–28). Rt 2.72 min (5 to 15% B over 0.5 min followed by 15 to 35% B over 3.5 min, λ = 214 nm); Calculated Mass [M+2H]2+: 1701.3, [M+3H]3+: 1134.5, [M+4H]4+: 851.2, [M+5H]5+: 681.1; Mass Found (ESI+): 1701.1 [M+2H]2+, 1134.4 [M+3H]3+, 851.0 [M+4H]4+, 681.0 [M+5H]5+.

Supplementary Fig. 17. Characterization of haemathrin-2 C-terminal diselenide dimer (29–59)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2 diselenide dimer (29–59); Rt 3.61 min (0 to 50% B over 5 min, λ = 215 nm). Calculated Mass [M+2H]2+: 1773.3, [2M+5H]5+: 1418.4, [M+3H]3+: 1182.5, [2M+7H]7+: 1013.5, [M+4H]4+: 887.2, [2M+9H]9+: 788.5, [M+5H]5+: 709.9, [2M+11H]11+: 645.3; Mass Found (ESI+); 1774.3 [M+2H]2+, 1419.4 [2M+5H]5+, 1182.9 [M+3H]3+, 1014.0 [2M+7H]7+, 887.3 [M+4H]4+, 788.7 [2M+9H]9+, 709.9 [M+5H]5+, 645.6 [2M+11H]11+.

Supplementary Fig. 18. Characterization of haemathrin-2 C-terminal diselenide dimer (29–59)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2 diselenide (29–59). Rt 2.48 min (5 to 15% B over 0.5 min followed by 15 to 35% B over 3.5 min, λ = 214 nm); Calculated Mass [2M+4H]4+: 1772.8, [2M+5H]5+: 1418.4, [2M+6H]6+: 1182.2, [2M+7H]7+: 1013.4, [2M+8H]8+: 886.9, [2M+9H]9+: 788.5, [2M+10H]10+: 709.7; Mass Found (ESI+): 1772.6 [2M+4H]4+, 1418.3 [2M+5H]5+, 1182.1 [2M+6H]6+, 1013.4 [2M+7H]7+, 887.0 [2M+8H]8+, 788.4 [2M+9H]9+, 709.8 [2M+10H]10+.

Supplementary Fig. 19. DSL preparation of haemathrin-1 (1–59)—Payne Lab, University of Sydney.

Analytical reverse-phase HPLC trace of the crude Haemathrin-1 A) post ligation and B) post deselenization.

Supplementary Fig. 20. Characterization of haemathrin-1 (1–59)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-1; Rt 3.41 min (0 to 50% B over 5 min, λ = 214 nm). Calculated Mass [M+4H]4+: 1673.5, [M+5H]5+: 1339.0, [M+6H]6+: 1116.0, [M+7H]7+: 956.7, [M+8H]8+: 837.3, [M+9H]9+: 744.3, [M+10H]10+: 670.0, [M+11H]11+: 609.2; Mass Found (ESI+): 1673.1 [M+4H]4+, 1338.8 [M+5H]5+, 1115.8 [M+6H]6+, 965.5 [M+7H]7+, 837.1 [M+8H]8+, 744.2 [M+9H]9+, 669.9 [M+10H]10+, 609.1 [M+11H]11+.

Supplementary Fig. 21. MALDI-TOF of haemathrin-1 (1–59)—Payne Lab, University of Sydney.

Calculated Mass for C279H445N91O101 [M+H]+: 6690.1; Mass Found: 6690.1 [M+H]+.

Supplementary Fig. 22. DSL preparation of haemathrin-1 (1–59)—Dep. Research Chemistry, Novo Nordisk.

Analytical reverse-phase HPLC (C18 BEH) of a) Crude DSL mixture during preparation of Haemathrin-1 after 10 min b) Crude deselenization mixture of Haemathrin-1 after 10 min (5% B over 0.5 min followed by 5 to 60% B over 3.5 min, λ = 214 nm).

Supplementary Fig. 23. Characterization of haemathrin-1 (1–59)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 BEH) and B) ESI mass envelope of reverse-phase HPLC purified Haemathrin-1. Rt 2.77 min (5 to 10% B over 0.5 min followed by 10 to 30% B over 3.5 min, λ = 214 nm); Calculated Mass [M+4H]4+: 1673.3, [M+5H]5+: 1338.9, [M+6H]6+: 1115.9, [M+7H]7+: 956.9, [M+8H]8+: 837.2, [M+9H]9+: 744.3, [M+10H]10+: 669.9, [M+11H]11+: 609.1; Mass Found (ESI+): 1673.2 [M+4H]4+, 1338.7 [M+5H]5+, 1115.8 [M+6H]6+, 956.5 [M+7H]7+, 837.1 [M+8H]8+, 744.2 [M+9H]9+, 669.9 [M+10H]10+, 609.1 [M+11H]11+.

Supplementary Fig. 24. DSL preparation of haemathrin-2 (1–59)—Payne Lab, University of Sydney.

Analytical reverse-phase HPLC trace of crude Haemathrin-2 post a) post ligation and b) post deselenization.

Supplementary Fig. 25. Characterization of haemathrin-2 (1–59)—Payne Lab, University of Sydney.

a) Analytical reverse-phase HPLC trace and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2; Rt 3.63 min (0 to 50% B over 5 min, λ = 214 nm). Calculated Mass [M+4H]4+: 1678.3, [M+5H]5+: 1342.8, [M+6H]6+: 1119.2, [M+7H]7+: 959.5, [M+8H]8+: 839.7, [M+9H]9+: 746.5, [M+10H]10+: 671.9, [M+11H]11+: 610.9; Mass Found (ESI+): 1677.9 [M+4H]4+, 1342.5 [M+5H]5+, 1118.9 [M+6H]6+, 959.3 [M+7H]7+, 839.5 [M+8H]8+, 746.3 [M+9H]9+, 671.7 [M+10H]10+, 610.8 [M+11H]11+. * Indicates artefact masses due to dimerization.

Supplementary Fig. 26. MALDI-TOF of haemathrin-2 (1–59)—Payne Lab, University of Sydney.

Calculated Mass for C280H446N88O104 [M+H]+: 6709.1; Mass Found: 6709.6 [M+H]+.

Supplementary Fig. 27. DSL preparation of haemathrin-2 (1–59)—Dep. Research Chemistry, Novo Nordisk.

Analytical reverse-phase HPLC (C18 BEH) of a) Crude DSL mixture during preparation of Haemathrin-2 after 10 min b) Crude deselenization mixture of Haemathrin-2 after 10 min (5% B over 0.5 min followed by 5 to 60% B over 3.5 min, λ = 214 nm).

Supplementary Fig. 28. Characterization of haemathrin-2 (1–59)—Dep. Research Chemistry, Novo Nordisk.

a) Analytical reverse-phase HPLC trace (C18 CSH) and b) ESI mass envelope of reverse-phase HPLC purified Haemathrin-2 Rt 1.78 min (5% B over 0.5 min followed by 5 to 60% B over 3.5 min, λ = 214 nm); Calculated Mass [M+4H]4+: 1678.1, [M+5H]5+: 1342.7, [M+6H]6+: 1119.0, [M+7H]7+: 959.3, [M+8H]8+: 839.5, [M+9H]9+: 746.4, [M+10H]10+: 671.8; Mass Found (ESI+): 1677.9 [M+4H]4+, 1342.5 [M+5H]5+, 1119.0 [M+6H]6+, 959.3 [M+7H]7+, 839.5 [M+8H]8+, 746.3 [M+9H]9+, 671.8 [M+10H]10+.

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Kulkarni, S.S., Watson, E.E., Premdjee, B. et al. Diselenide–selenoester ligation for chemical protein synthesis. Nat Protoc 14, 2229–2257 (2019). https://doi.org/10.1038/s41596-019-0180-4

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