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Peptide Synthesis: Methods and Protocols

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Peptide Therapeutics

Part of the book series: AAPS Advances in the Pharmaceutical Sciences Series ((AAPS,volume 47))

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Abstract

The development of the field of peptide synthesis has been integral in studying their physiological role in living systems as well as their use in developing therapeutics for many human diseases. From the early work of Emil Fischer to the advent of solid-phase peptide synthesis by R. Bruce Merrifield to today, peptide synthesis continues to evolve, allowing access to increasingly complex and previously unobtainable peptides in the lab. In this chapter, we give a brief history of peptide synthesis and outline some of the most used protecting groups, resins, linkers, and coupling methodologies. We end the chapter with a brief description of commonly used peptide modifications and give examples produced in the author’s laboratory.

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References

  • Al Musaimi O, de la Torre BG, Albericio F. Greening Fmoc/tBu solid-phase peptide synthesis. Green Chem. 2020;22:996–1018.

    Article  Google Scholar 

  • Bacsa B, Horvati K, Bosze S, Andreae F, Kappe CO. Solid-phase synthesis of difficult peptide sequences at elevated temperatures: a critical comparison of microwave and conventional heating technologies. J Org Chem. 2008;73:7532–42.

    Article  CAS  PubMed  Google Scholar 

  • Balanescu A, Radu E, Nat R, Regalia T, Bojinca V, Predescu V, Predeteanu D. Co-stimulatory and adhesion molecules of dendritic cells in rheumatoid arthritis. J Cell Mol Med. 2002;6:415–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baumann KH, Kiesel L, Kaufmann M, Bastert G, Runnebaum B. Characterization of binding sites for a GnRH-agonist (buserelin) in human breast cancer biopsies and their distribution in relation to tumor parameters. Breast Cancer Res Treat. 1993;25:37–46.

    Article  CAS  PubMed  Google Scholar 

  • Bayliss WM, Starling EH. On the causation of the so-called ‘peripheral reflex secretion’ of the pancreas. Proc R Soc Lond. 1902;69:352–3.

    Article  CAS  Google Scholar 

  • Beckers T, Bernd M, Kutscher B, Kuhne R, Hoffmann S, Reissmann T. Structure-function studies of linear and cyclized peptide antagonists of the GnRH receptor. Biochem Biophys Res Commun. 2001;289:653–63.

    Article  CAS  PubMed  Google Scholar 

  • Behrendt R, White P, Offer J. Advances in Fmoc solid-phase peptide synthesis. J Pept Sci. 2016;22:4–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Belwal VK, Datta D, Chaudhary N. The beta-turn-supporting motif in the polyglutamine binding peptide QBP1 is essential for inhibiting huntingtin aggregation. FEBS Lett. 2020;594:2894–903.

    Article  CAS  PubMed  Google Scholar 

  • Bergmann M, Zervas L. Ãœber ein allgemeines Verfahren der Peptid-Synthese. Ber Dtsch Chem Ges. 1932;65:1192–201.

    Article  Google Scholar 

  • Blackwell HE, Sadowsky JD, Howard RJ, Sampson JN, Chao JA, Steinmetz WE, O’Leary DJ, Grubbs RH. Ring-closing metathesis of Olefinic peptides: design, synthesis, and structural characterization of macrocyclic helical peptides. J Org Chem. 2001;66:5291–302.

    Article  CAS  PubMed  Google Scholar 

  • Bohmova E, Machova D, Pechar M, Pola R, Venclikova K, Janouskova O, Etrych T. Cell-penetrating peptides: a useful tool for the delivery of various cargoes into cells. Physiol Res. 2018;67:S267–S79.

    Article  CAS  PubMed  Google Scholar 

  • Carpino LA. Oxidative reactions of Hydrazines. II. Isophthalimides. New protective groups on nitrogen. J Am Chem Soc. 1957;79:98–101.

    Article  CAS  Google Scholar 

  • Carpino LA, Han GY. 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J Am Chem Soc. 1970;92:5748–9.

    Article  CAS  Google Scholar 

  • Chan W, White P. Fmoc solid phase peptide synthesis: a practical approach. Oxford: OUP; 1999.

    Book  Google Scholar 

  • Chatterjee B, Saha I, Raghothama S, Aravinda S, Rai R, Shamala N, Balaram P. Designed peptides with homochiral and heterochiral diproline templates as conformational constraints. Chemistry. 2008;14:6192–204.

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Flies DB. ‘Molecular mechanisms of T cell co-stimulation and co-inhibition’, nature reviews. Immunology. 2013;13:227–42.

    PubMed  Google Scholar 

  • Cheneval O, Schroeder CI, Durek T, Walsh P, Huang YH, Liras S, Price DA, Craik DJ. Fmoc-based synthesis of disulfide-rich cyclic peptides. J Org Chem. 2014;79:5538–44.

    Article  CAS  PubMed  Google Scholar 

  • Cheung JC, Kim Chiaw P, Deber CM, Bear CE. A novel method for monitoring the cytosolic delivery of peptide cargo. J Control Release. 2009;137:2–7.

    Article  CAS  PubMed  Google Scholar 

  • Colgrave ML, Korsinczky MJ, Clark RJ, Foley F, Craik DJ. Sunflower trypsin inhibitor-1, proteolytic studies on a trypsin inhibitor peptide and its analogs. Biopolymers. 2010;94:665–72.

    Article  CAS  PubMed  Google Scholar 

  • Davis SJ, Ikemizu S, Evans EJ, Fugger L, Bakker TR, van der Merwe PA. The nature of molecular recognition by T cells. Nat Immunol. 2003;4:217–24.

    Article  CAS  PubMed  Google Scholar 

  • De Marco R, Tolomelli A, Greco A, Gentilucci L. Controlled solid phase peptide bond formation using N-Carboxyanhydrides and PEG resins in water. ACS Sustain Chem Eng. 2013;1:566–9.

    Article  Google Scholar 

  • de Veer SJ, Kan MW, Craik DJ. Cyclotides: from structure to function. Chem Rev. 2019;119:12375–421.

    Article  PubMed  Google Scholar 

  • du Vigneaud V, Ressler C, Swan JM, Roberts CW, Katsoyannis PG. The synthesis of oxytocin. J Am Chem Soc. 1954;76:3115.

    Article  Google Scholar 

  • Eidne KA, Flanagan CA, Millar RP. Gonadotropin-releasing hormone binding sites in human breast carcinoma. Science. 1985;229:989–91.

    Article  CAS  PubMed  Google Scholar 

  • Fagundez C, Sellanes D, Serra G. Synthesis of cyclic peptides as potential anti-Malarials. ACS Comb Sci. 2018;20:212–9.

    Article  CAS  PubMed  Google Scholar 

  • Ferrazzano L, Corbisiero D, Martelli G, Tolomelli A, Viola A, Ricci A, Cabri W. Green solvent mixtures for solid-phase peptide synthesis: a Dimethylformamide-free highly efficient synthesis of pharmaceutical-grade peptides. ACS Sustain Chem Eng. 2019;7:12867–77.

    Article  CAS  Google Scholar 

  • Fields GB. Introduction to peptide synthesis. Curr Protoc Mol Biol. 2002; https://doi.org/10.1002/0471140864.ps1801s26. Chapter 11: Unit 11 15

  • Fischer E, Fourneau E. Ueber einige Derivate des Glykocolls. Ber Dtsch Chem Ges. 1901;34:2868–77.

    Article  Google Scholar 

  • Gentilucci L, De Marco R, Cerisoli L. Chemical modifications designed to improve peptide stability: incorporation of non-natural amino acids, pseudo-peptide bonds, and cyclization. Curr Pharm Des. 2010;16:3185–203.

    Article  CAS  PubMed  Google Scholar 

  • Gokhale A, Weldeghiorghis TK, Taneja V, Satyanarayanajois SD. Conformationally constrained peptides from CD2 to modulate protein-protein interactions between CD2 and CD58. J Med Chem. 2011;54:5307–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gokhale A, Kanthala S, Latendresse J, Taneja V, Satyanarayanajois S. Immunosuppression by co-stimulatory molecules: inhibition of CD2-CD48/CD58 interaction by peptides from CD2 to suppress progression of collagen-induced arthritis in mice. Chem Biol Drug Des. 2013;82:106–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gokhale AS, Sable R, Walker JD, McLaughlin L, Kousoulas KG, Jois SD. Inhibition of cell adhesion and immune responses in the mouse model of collagen-induced arthritis with a peptidomimetic that blocks CD2-CD58 interface interactions. Biopolymers. 2015;

    Google Scholar 

  • Guo H, Lu J, Hathaway H, Royce ME, Prossnitz ER, Miao Y. Synthesis and evaluation of novel gonadotropin-releasing hormone receptor-targeting peptides. Bioconjug Chem. 2011;22:1682–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guzman F, Gauna A, Roman T, Luna O, Alvarez C, Pareja-Barrueto C, Mercado L, Albericio F, Cardenas C. Tea bags for Fmoc solid-phase peptide synthesis: an example of circular economy. Molecules. 2021;26

    Google Scholar 

  • Hallbrink M, Floren A, Elmquist A, Pooga M, Bartfai T, Langel U. Cargo delivery kinetics of cell-penetrating peptides. Biochim Biophys Acta. 2001;1515:101–9.

    Article  CAS  PubMed  Google Scholar 

  • Hansen PR, Oddo A. Fmoc solid-phase peptide synthesis. Methods Mol Biol. 2015;1348:33–50.

    Article  CAS  PubMed  Google Scholar 

  • Harington CR, Mead TH. Synthesis of glutathione. Biochem J. 1935;29:1602–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoffmann K, Milech N, Juraja SM, Cunningham PT, Stone SR, Francis RW, Anastasas M, Hall CM, Heinrich T, Bogdawa HM, Winslow S, Scobie MN, Dewhurst RE, Florez L, Ong F, Kerfoot M, Champain D, Adams AM, Fletcher S, Viola HM, Hool LC, Connor T, Longville BAC, Tan YF, Kroeger K, Morath V, Weiss GA, Skerra A, Hopkins RM, Watt PM. A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery. Sci Rep. 2018;8:12538.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hojlys-Larsen KB, Jensen KJ. Solid-phase synthesis of phosphopeptides. Methods Mol Biol. 2013;1047:191–9.

    Article  PubMed  Google Scholar 

  • Isidro-Llobet A, Kenworthy MN, Mukherjee S, Kopach ME, Wegner K, Gallou F, Smith AG, Roschangar F. Sustainability challenges in peptide synthesis and purification: from R&D to production. J Org Chem. 2019;84:4615–28.

    Article  CAS  PubMed  Google Scholar 

  • Jad YE, Govender T, Kruger HG, El-Faham A, de la Torre BG, Albericio F. Green solid-phase peptide synthesis (GSPPS) 3. Green solvents for Fmoc removal in peptide chemistry. Org Process Res Dev. 2017;21:365–9.

    Article  CAS  Google Scholar 

  • Jaradat D’s MM. Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids. 2018;50:39–68.

    Article  CAS  PubMed  Google Scholar 

  • Jensen KJ. Solid-phase peptide synthesis: an introduction. Methods Mol Biol. 2013;1047:1–21.

    Article  CAS  PubMed  Google Scholar 

  • Kanthala SP, Liu YY, Singh S, Sable R, Pallerla S, Jois SD. A peptidomimetic with a chiral switch is an inhibitor of epidermal growth factor receptor heterodimerization. Oncotarget. 2017;8:74244–62.

    Article  PubMed  PubMed Central  Google Scholar 

  • Kantharaju S, Raghothama U, Raghavender S, Aravinda S, Shamala N, Balaram P. Conformations of heterochiral and homochiral proline-pseudoproline segments in peptides: context dependent cis-trans peptide bond isomerization. Biopolymers. 2009;92:405–16.

    Article  CAS  Google Scholar 

  • Kauffman WB, Fuselier T, He J, Wimley WC. Mechanism matters: a taxonomy of cell penetrating peptides. Trends Biochem Sci. 2015;40:749–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kremsmayr T, Muttenthaler M. Fmoc solid phase peptide synthesis of oxytocin and analogues. Methods Mol Biol. 2022;2384:175–99.

    Article  PubMed  Google Scholar 

  • Kumar A, Sharma A, de la Torre BG, Albericio F. Rhodiasolv PolarClean - a greener alternative in solid-phase peptide synthesis. Green Chem Lett Rev. 2021;14:545–50.

    Article  CAS  Google Scholar 

  • Lawrenson SB, Arav R, North M. The greening of peptide synthesis. Green Chem. 2017;19:1685–91.

    Article  CAS  Google Scholar 

  • Lesner A, Legowska A, Wysocka M, Rolka K. Sunflower trypsin inhibitor 1 as a molecular scaffold for drug discovery. Curr Pharm Des. 2011;17:4308–17.

    Article  CAS  PubMed  Google Scholar 

  • Li H, Aneja R, Chaiken I. Click chemistry in peptide-based drug design. Molecules. 2013;18:9797–817.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luna OF, Gomez J, Cardenas C, Albericio F, Marshall SH, Guzman F. Deprotection reagents in Fmoc solid phase peptide synthesis: moving away from Piperidine? Molecules. 2016;21

    Google Scholar 

  • Made V, Els-Heindl S, Beck-Sickinger AG. Automated solid-phase peptide synthesis to obtain therapeutic peptides. Beilstein J Org Chem. 2014;10:1197–212.

    Article  PubMed  PubMed Central  Google Scholar 

  • Matsuda S, Koyasu S. Mechanisms of action of cyclosporine. Immunopharmacology. 2000;47:119–25.

    Article  CAS  PubMed  Google Scholar 

  • Merrifield RB. Solid phase peptide synthesis. I. the synthesis of a Tetrapeptide. J Am Chem Soc. 1963;85:2149–54.

    Article  CAS  Google Scholar 

  • Millar RP. GnRHs and GnRH receptors. Anim Reprod Sci. 2005;88:5–28.

    Article  CAS  PubMed  Google Scholar 

  • Murray JK, Aral J, Miranda LP. Solid-phase peptide synthesis using microwave irradiation. Methods Mol Biol. 2011;716:73–88.

    Article  CAS  PubMed  Google Scholar 

  • Nagy A, Schally AV, Armatis P, Szepeshazi K, Halmos G, Kovacs M, Zarandi M, Groot K, Miyazaki M, Jungwirth A, Horvath J. Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent. Proc Natl Acad Sci U S A. 1996;93:7269–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Parajuli P, Sable R, Shrestha L, Dahal A, Gauthier T, Taneja V, Jois S. Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide. Chem Biol Drug Des. 2021;97:607–27.

    Article  CAS  PubMed  Google Scholar 

  • Patel SG, Sayers EJ, He L, Narayan R, Williams TL, Mills EM, Allemann RK, Luk LYP, Jones AT, Tsai YH. Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Sci Rep. 2019;9:6298.

    Article  PubMed  PubMed Central  Google Scholar 

  • Pawlas J, Rasmussen JH. ReGreen SPPS: enabling circular chemistry in environmentally sensible solid-phase peptide synthesis. Green Chem. 2019;21:5990–8.

    Article  CAS  Google Scholar 

  • Pawlas J, Nuijens T, Persson J, Svensson T, Schmidt M, Toplak A, Nilsson M, Rasmussen JH. Sustainable, cost-efficient manufacturing of therapeutic peptides using chemo-enzymatic peptide synthesis (CEPS). Green Chem. 2019;21:6451–67.

    Article  CAS  Google Scholar 

  • Schwyzer R, Sieber P. Total synthesis of adrenocorticotropic hormone. Nature. 1963;199:172–4.

    Article  CAS  PubMed  Google Scholar 

  • Sifferd RH, du Vigneaud V. A new synthesis of carnosine, with some observations on the splitting of the benzyl group from carbobenzoxy derivatives and from benzylthioethers. J Biol Chem. 1935;108:753–61.

    Article  CAS  Google Scholar 

  • Singh SS, Mattheolabakis G, Gu X, Withers S, Dahal A, Jois S. A grafted peptidomimetic for EGFR heterodimerization inhibition: implications in NSCLC models. Eur J Med Chem. 2021;216:113312.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Squires S, Christians E, Riedel M, Timothy D, Rodesch CK, Marvin J, Benjamin I. Effects of redox state on the efficient uptake of cell permeable peptide in mammalian cells. Open Biochem J. 2013;7:54–65.

    Article  PubMed  PubMed Central  Google Scholar 

  • Stawikowski M, Fields GB. Introduction to peptide synthesis. Curr Protoc Protein Sci. 2012; https://doi.org/10.1002/0471140864.ps1801s69. Chapter 18: Unit 18 1

  • Sugita M, Sugiyama S, Fujie T, Yoshikawa Y, Yanagisawa K, Ohue M, Akiyama Y. Large-scale membrane permeability prediction of cyclic peptides crossing a lipid bilayer based on enhanced sampling molecular dynamics simulations. J Chem Inf Model. 2021;61:3681–95.

    Article  CAS  PubMed  Google Scholar 

  • Taylor RE, Zahid M. Cell penetrating peptides, novel vectors for gene therapy. Pharmaceutics. 2020;12

    Google Scholar 

  • van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition. Annu Rev Immunol. 2003;21:659–84.

    Article  PubMed  Google Scholar 

  • Vanier GS. Microwave-assisted solid-phase peptide synthesis based on the Fmoc protecting group strategy (CEM). Methods Mol Biol. 2013;1047:235–49.

    Article  CAS  PubMed  Google Scholar 

  • Varnava KG, Sarojini V. Making solid-phase peptide synthesis greener: a review of the literature. Chem Asian J. 2019;14:1088–97.

    Article  CAS  PubMed  Google Scholar 

  • Walrant A, Bauza A, Girardet C, Alves ID, Lecomte S, Illien F, Cardon S, Chaianantakul N, Pallerla M, Burlina F, Frontera A, Sagan S. Ionpair-pi interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides. Biochim Biophys Acta Biomembr. 2020;1862:183098.

    Article  CAS  PubMed  Google Scholar 

  • Wegner K, Barnes D, Manzor K, Jardine A, Moran D. Evaluation of greener solvents for solid-phase peptide synthesis. Green Chem Lett Rev. 2021;14:153–64.

    Article  CAS  Google Scholar 

  • Winkler DFH. Automated solid-phase peptide synthesis. Methods Mol Biol. 2020;2103:59–94.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Agricultural Chemistry, LSU AgCenter, Baton Rouge, LA, USA

    Ted Gauthier & Dong Liu

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  1. Ted Gauthier
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  2. Dong Liu
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Correspondence to Ted Gauthier .

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Editors and Affiliations

  1. School of Basic Pharmaceutical and Toxicological Sciences, University of Louisiana at Monroe, College of Pharmacy, Monroe, LA, USA

    Seetharama D. Jois

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Gauthier, T., Liu, D. (2022). Peptide Synthesis: Methods and Protocols. In: Jois, S.D. (eds) Peptide Therapeutics. AAPS Advances in the Pharmaceutical Sciences Series, vol 47. Springer, Cham. https://doi.org/10.1007/978-3-031-04544-8_2

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