Lara Malins Baran Group Meeting Highlights in Peptide and Protein Synthesis 04/23/16 Peptides as Therapeutic Leads: NH2 The Golden Oldies: H O N • ~100 therapeutic peptides currently on market O N • Octreotide, Goselerin, Leuprolide - top sellers O H • Approval rate for peptides (20%) vs. for small molecules (10%) S HN "My entire yearning is directed toward the synthesis of • Anticipated global market of US$23.7 billion by 2020 S O HN O NH the first enzyme. If its preparation falls into my lap with OH H H N N the synthesis of a natural protein material, I consider -EuPA Open Proteomics 2014, 4, 58 N H my mission fulfilled." O O HO OH Peptides in the news: - Emil Fischer, 1905 Octreotide NH2 (letter to Adolph von Baeyer) (Novartis) OH ...Rugby league player suspended after admitting H O Theodor Curtius Emil Fischer O N to the "use and trafficking of peptides" OH O O O H H N NH N N H 2 (August 2013) N N N H H Theodor Curtius O NH O O HN N O J. Prakt. Chemie 1882, 26, 145 NH O O O NH NH O O H N O 2 H N + HN NH2 2 OAg 2 Ph Cl Ph N OH + Ph OH HN O H O Goselerin + 2AgCl NH (AstraZeneca) Acyl azide coupling "peptides can assist you during your journey to health J. Prakt. Chemie 1904, 70, 57 and well-being" (https://peptidesdirect.com.au) O O O O O H H H Ph N N3 N N Ph N H2N OEt N OEt Scope of this meeting: (aka Lara's peptide mixtape): H O H O O A O Peptides: The Greatest Hits • Brief historical perspective on peptide synthesis, outlining 1) H2NNH2 challenges and discoveries in the early years (1882-1960s) 2) HNO2 • Development of solid-phase peptide synthesis (SPPS) • Advances in coupling reagents and protecting groups • Chemoselective ligation chemistry for the synthesis of O O O O O H H H A H H large peptides and proteins Ph N N N Ph N N N OEt N • Perspectives and unmet challenges N N3 H H H • A collection of some personal favorites! Peptides: Greatest Hits Collection O O O O O (1882-present) pentaglycine peptide Timeline: Cbz SPPS ligation Emil Fischer 1882 1932 1963 1992 present Ber. Dtsch. Chem. Ges. 1901, 34, 2868 O O H conc. HCl N HN NH Cl glycylglycine 1901 1953 1970 1994 H3N OH " O dipeptide oxytocin Fmoc NCL O Gly-Gly The PepTides - "Love Question Mark" (2013) Acid chlorides in peptide synthesis and the #-bromoacyl method Ber. Dtsch. Chem. Ges. 1905, 38, 605 Topics not covered: www.thepeptides.com OEt • Non-amide bond forming ligation methods (e.g. bioconjugation, see Sella GM, May 2014 - ADCs) H N O 2 base/ • Biological expression or semi-synthetic approaches (e.g. expressed protein ligation, unnatural O O O amino acid incorporation - cf. P. Schultz) Br Cl H NH3 H N N OEt N OH Unnatural amino acid/peptide derivatives ( -peptides, peptoids, peptidomimetics) H • ! O Br N H2N N H H • Post-translational modifications (glycoslyation, sulfation, phosphorylation) and the synthesis of post- O O O O translationally-modified peptides or proteins "masked" Leu amine leucylglycylglycine Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16 Octadecapeptide - Fischer Unidirectional chain growth: Ber. Dtsch. Chem. Ges. 1907, 40, 1754 C-to-N: O R' OH O O R O R''O N 3 O R O H H H H H H N O 3 N N Leu-(Gly)3-Leu-(Gly)3-Leu-(Gly)8-Gly H N OMe R''O N N Br N Cl 2 2 1 N 2 OMe H H 1 O O O Boc, Fmoc, Cbz O O (racemization-suppressing R' Practical Limitations: urethane group) N C lack of readily cleavable N-terminal protecting groups chain growth • N-to-C: • limited access to enantiopure amino acids O H2N O R OMe O R O H 1 H H Essential protecting groups (urethanes) R''O N 3 OH R''O N 3 N N 2 N 2 OMe H H 1 O O O O O O O R' R' R R R X O N O N O N N C H H H N-acyl group chain growth epimerization at C-2 Boc racemization-prone Cbz (Z) Fmoc Carpino Bergmann and Zervas JACS 1957, 79, 4427 Carpino Vincent du Vigneaud Ber. Dtsch. Chem. Ges. McKay and Albertson JACS 1970, 92, 5748 1932, 65, 1192 • Head of Biochemistry - Cornell University Medical College JACS 1957, 79, 4686 • Studied the biochemical importance of sulfur-containing compounds (insulin, penicillin, oxytocin, vasopressin) H2/Pd Deprotection Na/NH (l) TFA base (amines) • First total chemical synthesis of a bioactive peptide hormone Conditions: 3 HBr/AcOH HCl NH3 (l) • Nobel Prize in Chemistry (1955) A convenient finding... Total Synthesis of Oxytocin J. Am. Chem. Soc. 1953, 75, 4879 (Vigneaud) R R O H2NR' O R O H2NR' O R • Prepared using a 3-fragment condensation approach du Vigneaud Cl NHR' Cl NHR' Ph N Ph N Ph O N Ph O N • Coupling methods: acid chlorides, anhydrides, H H H H pyrophosphite method OH O O Cbz O O benzoyl racemization configurationally • Purified by counter-current distribution (CCD) III stable • No HPLC, mass spec, high resolution NMR! O • Overall yield <<1% Ber. Dtsch. Chem. Ges. 1932, 65, 1192 H2N O N H Oxytocin HN Racemization through oxazolone formation (major) S S R R R O HN O II O H OR* H R N N N N PGHN OR* N R' R' O N O H H N O O O O O O H R' O N O O PGHN PGHN H2N N O R' O H PGHN O NH2 I NH2 Racemization through enolization (minor) R Improved oxytocin synthesis published in 1959: stepwise, p-nitrophenyl ester couplings, C-to-N O R O R N CCD apparatus OR* R' (invented by Lyman Craig) elongation, 38% overall yield PGHN PGHN OR* OH N N O J. Am. Chem. Soc. 1959, 81, 5688 (Vigneaud) H H PGHN R' O R' O 2 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16 Bruce Merrifield and Solid-Phase Peptide Synthesis (SPPS) Common linkers: NH Biochemist by training, moved to Rockefeller Institute for Medical 2 • O OMe Research (1949) Linker = R • Work on the structure of peptide growth factors required the synthesis of a Cl pentapeptide: Rink amide linker "my overall yield of pentapeptide was 7%, and it took me 11 months. OMe Certainly, an experienced peptide chemist would have done better, but not O without considerable effort." Cl HN OH Trityl linker (R = H) Merrifield 2-Cl trityl linker (R = Cl) 29 May 1959 - Merrifield records concept of SPPS in his lab book (chloromethyl) PAM linker (4-hydroxymethyl-phenylacetamidomethyl) 1984 - Awarded the Nobel Prize for SPPS Merrifield Solid-phase peptide synthesis (SPPS) = solid support (resin) OH OH N N Deprotect H H O = N-terminal amino O N-terminus O Linker O Linker protecting group Linker N H2N Load resin H couple O O amino acid = side-chain protecting groups i) Deprotect (global) O ii) Cleave from resin H Peptide 1 Linker N O Linker H2N Peptide 1 OH N For development of solid-phase iii) HPLC purify Repeat: deprotect H O O oligonucleotide synthesis, see: and couple Synthetic peptide product J. Am. Chem. Soc. 1966, 88, 5319 Seminal Paper J. Am. Chem. Soc. 1963, 85, 2149 (Merrifield) Common Resins: 1) - polystyrene +1-2% divinylbenzene (DVB) - ChemMatrix polyethylene glycol (PEG): cross-link: O 1) Cbz removal: CbzHN NHEt3 O 30% HBr in AcOH Cl Merrifield linker O (2-4 h) e.g. CbzHN Cl O 2) O O H N O O 2) Et3N/DMF 2 n NH2 O NHEt3 O H N O 1) OH 2 O NH2 SPPS CbzHN n then O O O O H N O NaOH/EtOH H 2 O NH2 N DIC, DMF, rt, 18 h H2N n CbzHN O O DVB O 2) Ac2O/Et3N cross-link more polar resin rt, 2 h decreases hydrophobic aggregation (PCT Int Appl 2005, WO 2005012277 A1) O O H A reviewer's thoughts: the approach is "a H N N OH Properties to consider: 2 N N travesty...not chemistry at all, a concept which H H Resin loading: 0.2 - 1.5 mmol/gram O O should be surpressed by the community." • (number of functionalizable sites) Cl Leu-Ala-Gly-Val (8% yield of Biopolymers 2008, 90, 175 (Mitchell) • "Swellability": determines appropriate solvents Merrifield resin purified compound) 3 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16 Automation Improvements in Coupling Reagents "Automated Synthesis of Peptides" Reviews: Chem. Rev. 2011, 111 , 6557 (Albericio); Tetrahedron 2004, 60, 2447 (Han) Science 1965, 150, 178 HOBt in peptide synthesis Chem. Ber. 1970, 103, 788 (König) - 965 citations N Synthesis of an Enzyme with Ribonuclease A (RNase) Activity N N N J. Am. Chem. Soc. 1969, 91, 501 (Merrifield) PF N PF6 PF6 N N 6 N N "Assembly of the 124 amino acid residues into the protected, N O Br N NMe2 P N P N resin-bound straight-chain precursor of RNase required 369 OH BOP O P N N chemical reactions and 11,931 steps of the automated peptide NMe2 synthesis machine." HOBt Me2N • N-terminal Boc protecting groups Racemization Tet. Lett. 1975, 16, 1219 PyBOP PyBrOP • DCC as the coupling reagent - JACS 1955, 77, 1067 (Sheehan) suppressor (Castro) Tetrahedron 1991, 47, 259 (Coste) Merrifield and his peptide **Final product had enzymatic activity (though less than native RNase) synthesis machine HOAt as a peptide coupling additive J.