Highlights in Peptide and Protein Synthesis 04/23/16

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 N-terminus O = N-terminal amino O 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. Am. Chem. Soc. 1993, 115 , 4397 (Carpino) - 1367 citations

N O Boc SPPS (Boc/benzyl) t Fmoc SPPS (Fmoc/ Bu) N HO N N N-terminal Fmoc N-terminal Boc OH O piperidine labile O O HOXt/EDC TFA labile HOAt NH N O O side-chain tBu HOAt: complete at 22 h side-chain O or Trt benzyl PGs N HOBt: trace product O O TFA labile HF labile O N N O H N N O N O Resin N O H via: O Resin TFA labile N HF labile OH O HOBt X Pros: Pros: • Optimizable to 50-60 residues (less • mild and safe (no HF required) O O O O aggregation prone) • spectrophotometric quantification of couplings conditions H • Stable thioester linkages OH + H2N N N OMe N OMe Cons: H Cons: • More aggregation prone H O O • HF safety hazard • Incompatible with base sensitive • Limited FG compatibility substrates (e.g. thioesters) N coupling conditions DL-isomer (%) N Fmoc Spectrophotometric Quantification HOAt: N N DCC, 24 h 61.5% O HOBt, DCC, 24 h 41.9% • superior leaving group O • neighboring group effect HOAt, DCC, 24 h 14.4% H HOAt, DCC, DCM, 24 h < 1-2% H N R O N N R R H R R O N H2N H -CO2 fast Oxyma: A Safer Alternative K-Oxyma: for highly acid-labile resins piperidine-fulvene adduct Chem. Eur. J. 2009, 15, 9394 (Albericio) Eur. J. Org. Chem. 2013, 6372 (Albericio) != 7800 cm-1 (" = 301 nm) NC COOEt NC COOEt • Slightly more reactive than HOAt • non-acidic alternative N • Non-explosive (unlike HOBt, HOAt) N to Oxyma OH O K 4 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

"Alphabet Soup": Why are there so many coupling reagents? Synthesis of 5-N-acetylardeemin O J. Am. Chem. Soc. 1999, 121, 11953 (Danishefsky) O H • Coupling of bulky (and/or non-proteinogenic) amino acids N C15b H2N Me OH H-Ala-CO2Me • Considerations for removal of coupling reagent byproducts OH DCC/DMAP • Enhancing rate of amide bond formation O -aminoisubutyric acid DCC/HOBt H • Minimizing racemization of activated acid ! H O (Aib) N-Me-valine N BOP-Cl N NH N OH partial NBoc O N N N racemization Me N Boc H Case Studies: O H (C15b or C8) Boc Boc MeO2C Synthesis of (-)-Mirabazole C - Application of PyBroP O J. Org. Chem. 1992, 57, 5566 (Heathcock) cyanuric fluoride C8 pyridine 1) HBr, HOAc o DCM, -15 C NaHCO3, H2O BnS BnS BnS 2) A, PyBroP PyBroP, DIEA DIEA, DMAP DCM Me Me DMAP, DCM, 4 h Me O DCM, 24 h See also: TFFH as a reagent for 71% (2 steps) H O OH + OMe N acid-fluoride couplings: H Me CbzHN H3N 90% CbzHN OMe 60% JACS 1995, 117 , 5401 (Carpino) + Cl O O O Me F H2N CO2Me A N BnS N N N Boc TFFH Boc H PF6 Acid chloride, BOP, BOP-Cl, DPPA, DCC were unsuccessful in coupling A F

BnS BnS Me Me O O O Aib couplings with the Aziridine method H O H H R O CbzHN N Helv. Chim. Acta 1987, 70, 102 (Heimgartner) H N N SBn N OMe N N N H Helv. Chim. Acta 1990, 73, 13 CbzHN NR H H Me O Me 2 O Me O Me O BnS NR BnS BnS BnS 2 HN 1) Na, NH3 N R R NR R O 63% 2) NH4Cl Et O, rt, 0.5-6 h 2 H 2 O N 3) TiCl4, DCM OH CbzHN CbzHN NR NiO2, benzene CbzHN 2 N N reflux, 6 h N N O O O S S N N S 60% N N S Me Me Me Me S S S Choosing a coupling reagent: where to begin? Me S Me (-)-Mirabazole C Start

Synthesis of Palau'amine solid-phase solution-phase J. Am. Chem. Soc. 2011, 133, 14710

NH2 aniline? NH2 Cl NH Cl NH NH bulky AA, NH base H3N H N H difficult general 3 NH sensitive? conds general bulky AA, N 2 coupling conds difficult N EDC/HOBt coupling acid OH NH chloride H3N DIC/HOBt PyAOP PyBOP HOBt/DIC or DCC HOAt/DIC NH NH O HBTU Other attempts: HN N DIC/oxyma HATU or EDC acid fluoride HOBt/DIC HATU, HBTU, TCTU, DPPA, HOAt/DIC (water-soluble) NH 2 T3P, EDC, DIC, DCC, BOP, O PyBrOP BOP-Cl, PyAOP, PyBOP, PyBrOP, cyanuric chloride 5 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

A Few Notable Protecting Groups JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.) Review: Chem. Rev. 2009, 109, 2455 (Albericio) Alternatives to Fmoc J. Am. Chem. Soc. 1997, 119, 9915 (Carpino) N NH

O NH O (1 equiv.) O2S N O NH DCM, rt, 3 h O N O N O S H CH2 O O N O O2 S O HN Cl O O2 O N -CO2 S O O Bsmoc Michael 2 N H N Cl O H addition 2 H2N SO2 O H2N NH2 O NH O N PEG PEG 2'-paclitaxel OH NH2 glycinate O Bsmoc removal O O O EDC, DMAP 2% in DCM only O DCM, rt, 12 h Leu-Leu-Leu O O N O 82% (2 steps) H SO2 PEG N H H Me N Selective removal of Bsmoc or Fm/Fmoc Fm removal 10% in DCM only Perspectives on Protein Synthesis Relative sensitivity to piperidine: H Limitations of Solid- and Solution-phase couplings: • SPPS (and subsequent coupling reagent and PG advances) greatly improved the effiency of O > O peptide preparation but did not substantially expand the size of accessible targets S O S O > O O • On-resin aggregation generally limits routine SPPS to ~50 amino acids 2 O2 S O • Solution-phase fragment condensations (using protected peptides) are plaqued by the poor O2 $!!" solubility of protected peptides !-Nsmoc Bsmoc "-Nsmoc JOC 2007, 72, 1729 JACS 1997, 119, 9915 JOC 2007, 72, 1729 JOC 1999, 64, 4324 $!!"

Price comparison (Alfa Aesar) 5 g Fmoc-Phe-OH: $16.65 5 g Bsmoc-Phe-OH: $107.26

Application of Bsmoc protection !Protein Functional Domains JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.) #!!" #!!" O O BsmocHN OH 56")*"78&4)"79&0:;" O O 56")*"78&4)"79&0:;" CH OH 12%&'%/3"0&%/-",45% Smallest Proteins O NH H C 3 EDC, DMAP O NH %&'(")*"+),-.(./0("123&4"" O 3 O %&'(")*"+),-.(./0("123&4"" o !"#$%&'%(&)*($(+,$%-./"0% CH3 DCM, 10 C to rt, 1.5 h Cbz- O O CH3 SPPS Chemical Ligation OH O O H O HO !" !" O O O !"#$%!"#&%!"!'%!"!(%!"'#%!"'$%!"'&%!")'%!")(%!"$#%!"$$%!"$&%!"*'%!"*(%!"(#%!"($%!"(&%!"+'%!"+(%!"&#%!"&$%!"&&%!""'%!""(%'###%'##$% O !"#$%!"#&%!"!'%!"!(%1906!"'#%!"'$% !"'&%!")'%!")(%!"$#%1932!"$$% !"$&%!"*'%!"*(%!"(#%!"($%1962!"(&% !"+'%!"+(%!"&#%!"&$%!"&&%1992!""'%!""(% '###%'##$% Taxol N <(3=" O O H <(3=" SO2 Annu. Rev. Biochem. 2000 69, 923 6 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

Chemical Ligation Optimized templates - 4,6-dibenzofurans OH OH J. Org. Chem. 1986, 51, 1829 (Kemp) NH NH 3 3 NH X NH3 3 X OOC Chemoselective OOC ligation + Z o DMSO, 25 C Z SH • unprotected peptides SH O acyl shift O • aqueous solution O S COO COO S HO S • physiological pH O S SH SH HO mutually reactive HO R H N functional groups 2 HN CO Me Annu. Rev. Biochem. 2000 69, 923 2 CO Me X = H, Cl, Br, NO2 R 2 Chem. Soc. Rev. 2009, 38, 338 Z = H, Cl O

Amide bonds through acyl transfer ring substituents acyloxy k1 /kH Effective -1 Justus Liebigs Ann. Chem. 1953, 583, 129 (Wieland) X Z R k1(s ) t1/2 (rel rate) molarity (EM)*

-5 H H CH3 9.7 x 10 2 h 1 (ref) 4.6 Na2CO3 Br H CH 4.8 x 10-4 24 min 4.9 HS 3 HS H2O S S to N CH 1.1 x 10-3 10 min H N Cl H 3 11 4.5 0 oC 2 acyl shift O -5 SPh + H H Z-Ala 7.6 x 10 2.5 h 1 (ref) 5.1 O O H2N O -4 H3N H3N O N Br H Z-Ala 7.8 x 10 15 min 10 - PhSH H2N H -3 Cl O O O Cl H Z-Ala 1.2 x 10 10 min 15 5.7 O -1 NO2 H3 Z-Ala 2.4 x 10 2.8 s 3 3200 3 8.6 3 H Cl Z-Ala 3.5 x 10-3 3.3 min 45 4.9 Prior thiol capture Cl2 Cl2 Z-Ala2 7.6 x 10-3 1.5 min2 1002 2.72 Tetrahedron Lett. 1981, 22, 181 (Kemp) *EM determined from the ratio of k1 to k2 (k2 = rate constant for intermolecular aminolysis of each 1- Tetrahedron Lett. 1981, 22, 185 (Kemp) B and/or 3-functionalized 4-(acyloxy)dibenzofuran template with H-Cys(Bn)-OEt in DMSO at 25 oC

Total Synthesis of BPTI - Basic Pancreatic Trypsin Inhibitor (30-58) O O O H2N PG S J. Org. Chem. 1989, 54, 2803 (Kemp) A O S Template A O SH Thiol A O S H Cys(Acm) O Cys(Scm) activation capture N Template Template Template (deprotection) O S O to N O S PG O SH acyl S transfer MRT O SH BocHN O + PG H N B GGA HO S 2 O H2N PG B O HO S O 1) HFIP/H O (thiol capture) HS 2 R' = A N 2) DMSO (acyl transfer) Template O Release H S 4) Dnp-F, DIEA (Cys protection) 3) PEt3, DCM/HFIP R n = 0, 1 template OH S A N B 5) TFA/thioanisole H (template cleavage) H (reduction) (remove non-Cys PGs) N Template 6) Scm-Cl (convert Acm to Scm) HS Tetrahedron Lett. 1981, 22, 181 (Kemp) O S PG O O OAc O OAc GGA Me MRT N Me concentration independent BocHN • O H O N to O acyl O to N acyl transfer O transfer O OMe (intramolecular) O S O OMe DnpS Repeat 1-6 for OH S • effective local conc. of O S t1/2 = 2.7 h (DMSO) S 2 more ligations S intramolecular amine = 0.6 M S O 28 h (DMF) H BPTI 30-58 O GGA H N N MRT N 2 H2N H 29 residue peptide CO2Et O CO2Et O O 7 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

Thioester ligation - "Backbone-engineered" proteins Total Synthesis of Interleukin-8: thiol additive* Science 1992, 256, 221 (Kent) Science 1994, 266, 776 (Kent) 6 M Gn•HCl/ HS O O H 0.1 M Na2HPO4 H N pH = 4.3 N Br B A S B SH 6 M Gn HCl/ A SH + H O HS • SH phosphate buffer O O N 33 H IL-8 (1-32) S pH = 7.6 thioester linkage + N H2N IL-8 (35-72) 34 23 oC, 48-72 h Synthesis of HIV-1 protease: O SH O O N HO H HS S SH O SH H2N 51 H H O N 33 N automated Folded IL-8 IL-8 (1-32) N 34 IL-8 (35-72) automated H Boc-SPPS O Boc-SPPS SH O O O *Thiol additive serves as a reductant to prevent disulfide formation and to reverse unproductive N thioesterification of internal Cys residues H Br S HIV-1 PR(53-99) O HIV-1 PR(1-50) N 51 52 H O Rate enhancement with thiol additives HF HF J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent)

O Transthio- O O esterification HS SH Br A S + + HIV-1 PR(1-50) N 51 HIV-1 PR(53-99) OH A S H O 52 HS Benzyl thioester Thiophenyl thioester [peptides] = 4 mM 6 M Gn•HCl/0.1 M Na2HPO4 easier to handle more reactive pH = 4.3, 48 h (HPLC stable) in ligations

Comparative synthesis of Barnase (1-110): O J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent) S HIV-1 PR(1-50) N 51 HIV-1 PR(53-99) OH H 52 O HS H O N 48 H assembled HIV-1 PR dimer exhibits enzymatic activity barnase (1-47) S + N H2N 49 barnase (50-110)* O Native Chemical Ligation (NCL) Science 1994, 266, 776 (Kent) HS 6 M Gn HCl/0.1 M Na HPO 4 vol% or • 2 4 pH = 7.5 HS A S HS O Transthio- S!N H esterification N O acyl shift + H A SR H2N B N HS H N fast O O slow 2 B H H 48 thioester N-terminal Cys O N N barnase (1-47) N 49 barnase (50-110)* peptide peptide H O HS O • Construction of native amide bonds in aqueous media H For benzylthiol: For PhSH: Completely chemoselective (alcohols, amines, acids, internal N • A N B < 25% complete at t = 7 h complete at t = 7 h thiols, etc. are all tolerated) - allows use of unprotected peptides H O 8 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

Fine-tuning with thiol additives Convergent Synthesis of Human Lysozyme J. Am. Chem. Soc. 2006, 128, 6640 (Kent) PNAS 2007, 104, 4846 (Kent) HS R 130 amino acids - 4 fragments, 3 ligations Acm HO SR' O KVFERCELAR TLKRLGMDGY RGISLANWMC S Acm HS O HS R' pKa ! 9 R R' inefficient LG LAKWESGYNT RATNYNAGDR STDYGIFQIN R A S SRYWCNDGKT PGAVNACHLS CSALLQDNIA 66-94 S R 96-130 A S in ligation N H2N A S DAVACAKRVV RDPQGIRAWV AWRNRCQNRD H O O O pKa < 4 - inefficient at VRQYVQGCGV HS ligation MPAA transthioesterification Acm B Ideal thiol additive: 6 < pKa < 9 H N 2 1-29 S HS Acm O OH S Acm O O O 96-130 N 66-94 N H H HS O O kinetically-controlled Thz HS ligation (KCL) 31-64 S R MeONH , pH = 4 HS H N removal 2 O HS NH2 2 HO O O O HO HS Acm HS Acm HS Acm 4-mercaptophenylacetic acid O O HS (MPAA) S R 1-29 31-64 HS N 66-94 96-130 stable solid H H2N N • O O H HS N • water-soluble O O • rapid ligation • no epimerization 1) ligation: MPAA • minimal stench! 2) Acm removal: AgOAc, DTT HS HS HS O O O 66-94 96-130 31-64 N N 1-29 N H H H O O O Kinetically-controlled ligations Angew. Chem. Int. Ed. 2006, 45, 3985 (Kent) One-pot KCL to homogeneous EPO Angew. Chem. Int. Ed. 2012, 51, 11576 (Danishefsky) "At last..." See also: Science 2013, 342, 1357 HS HS O O Acm O HS Acm S NH C A B 2 H2N 1-29 S S H2N OH HO O 31-78 SEt HO O latent O H2N alkyl thioester OH O O O = ligation #1 ligation #2 AcHN (after activation) KCL, 5 h; then: MPAA, CO H HO O 2 - Exploiting thioester reactivity allows for Iterative ligations Acm HS One-pot HO OH HO2C HO OH HO O HO Convergent Synthesis of Human Lysozyme kinetically-controlled O O PNAS 2007, 104, 4846 (Kent) 80-166 ligation AcHN O O O H2N HO NHAc AcHN 130 amino acids - 4 fragments, 3 ligations O HS OH OH HS HS O O O O HS = HO O HS Acm Acm O 66-94 96-130 Acm O HO HO 31-64 N N O NHAc NHAc 1-29 N H H H O O 31-78 80-166 O 1-29 N N native chemical native chemical H kinetically-controlled H O O ligation ligation ligation EPO (1-166) precursor 9 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

Thia-zip reaction J. Am. Chem. Soc. 1999, 121, 4316 (Tam) HS HN HS HN HS HN HS HN HN HS Method for the synthesis of cysteine-rich cyclic peptides

S S cyclopsychotride MeO MeO OMe S S J. Nat. Prod. 1994, 57, 1619 OMe S S NO2 OMe CSCKSKVCYKNSIPCGESCVFIPCTVTALLG - one of the largest known cyclic peptides (31 AAs) Org. Lett. 2000, 2, 23 Bioorg. Med. Chem. Lett. 2002, 12, 1963 Angew. Chem. Int. Ed. - 6 cysteine residues and 3 disulfide bonds Org. Lett. 2001, 3, 1403 J. Am. Chem. Soc. 2002, 124, 4642 2015, 54, 15055

Side-chain HS Auxiliary-based HS HS S O HS SH SH SH SH SH SR SH SH SH SH Cys surrogates O H2N A RHN A H2N H2N

HO OH OH OH pH = 7.5 thiolactone HS = O HO O H SH (8 M urea) exchange O HO O N SH HO HO O HO S NH HN NH O acyl shifts HS SH S O O SH SH SH O HS O NH2 HS O HS SH SH SH (20 h - 24 h) H2N JACS 2006, 128, 5626 JACS 2006, 128, 15026 Chemistry 2007, 13, 5670 JACS 2007, 129, 7690 OH O S to N blocking terminal irreversible blocking internal HO O O acyl shift thiol prevents thiol still leads O O lactam formation O NH NH to lactam formation NHAc O O SH HS SH O HS O HS O HS Angew. Chem. Int. Ed. [O] Chem. Commun. 2008, 10, 1229 N DMSO 2007, 46, 5975 H cyclopsychotride disulfide SH SH SH Thia-zip cyclization is 200x faster Ligation-Desulfurization formation than unassisted cyclization HS Pd/Al2O3, H2 O O H Reductive H N N What about Cys-free peptides and proteins? A N B A N B H VA-044, TCEP, tBuSH H Relative abundance (Cys) = 1.7% O O N-terminal R Radical Cys HS Auxiliary-based A Ala HN Reductive: JACS 2001, 123, 526 (Dawson) - 373 citations Cys surrogates HS O Radical: ACIE 2007, 46, 9248 (Danishefsky) - 311 citations H2N A HN N HS H HO C = N N 2 N N P H HO2C CO2H HN N HN VA-044 TCEP HN HS R HS HS HS Thiol amino acids: N H O N R e.g. H R OMe 2 TrtS O HS N O RS RS Reviews: R = H, OMe R = H, OMe OtBu Curr. Opin. Chem. Biol. 2014, 22, 70 OH JACS 1996, 118 , 5891 Bioorg. Med. Chem. 2001, 9, 2323 OH N OH Aust. J. Chem. 2015, 68, 521 Tet. Lett. 2003, 44, 6059 BocHN BocHN Tet. Lett. 2001, 42, 1831 Bioorg. Med. Chem. O R O O PNAS 2001, 98, 6554 2004, 12, 2714 10 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

Staudinger ligation Staudinger Ring-Closure (Medium-Sized Lactams) Review: Angew. Chem. Int. Ed. 2004, 43, 3106 Angew. Chem. Int. Ed. 2003, 42, 4373 (Maarseveen) NH2 "Cell-surface engineering" NH2 Science 2000, 287, 2007 (Bertozzi) Cl NH NH Cl NH NH N N3 DABCO 3 H N NH2 OH MeO O MeO O 60 oC MeO O N sample N NH PPh2 N3 sample S PPh2 PPh2 20% N3 NH NH O HN N -N Ph2P 2 aza ylide NH2 O O O O O BH3 O OH J. Am. Chem. Soc. 2011, 133, 14710

• "Bioorthogonal" tagging • aqueous media KAHA (Ketoacid-hydroxylamine) ligations • on the surface of living cells Angew. Chem. Int. Ed. 2006, 45, 1248 (Bode) • inside living animals fast (see: Nature 2004, 430, 873) O R DMF, 40 oC R H O H HO (50 - 100 mM) H N sample OH + N N O A N B A N B O MeO N sample H -CO2 H O O O O -H2O PPh2 PPh2 J. Am. Chem. Soc. 2008, 130, 4253 (Bode)

NC modification of cell O O O O S surface glycans Br R HBTU, DIEA R S Oxone R O DCM, rt 1:1 DMF/H2O OH BocHN BocHN CN BocHN OH O O "Traceless" Staudinger Peptide Ligation O cyanosulfur ylide Org. Lett. 2000, 2, 2141 (Bertozzi) Org. Lett. 2001, 3, 9 (Raines) Org. Lett. 2000, 2, 1939 (Raines) J. Am. Chem. Soc. 2007 129, 11421 (Raines) Solid-phase approach Org. Biomol. Chem. 2009, 7, 2259 (Bode) O O

A S PPh2 + N3 B A S PPh2 PF6 H2C-CN Fmoc-Phe-OH H -N2 1:2 MeCN/PhMe N "traceless" auxiliary N B HN HATU, HOAt O HN 15 h, 60 oC DIEA, DMF O O O O O O SH FmocHN S HS PPh2 Rink resin PPh2 CN O S Fmoc- HO PPh O H O S PF6 SPPS 2 2 B TFA B A N OH A N CN H Ph2P PPh H N 2 PPh2 NH2 Oxone O N Ph2P SH O HS H DMF/H O O O peptide N OH 2 O H HS S Ar = O peptide N PAr 2 N CN H2O soluble 11 Baran Group Meeting Lara Malins Highlights in Peptide and Protein Synthesis 04/23/16

KAHA: Increased reactivity through ring-strain "Rethinking amide bond synthesis" Nature Chem. 2015, 7, 668 (Bode) Review: Nature 2011, 480, 471 (Bode) O 0.1 - 5 mM HO CuI (1 mol%) O 20-25 oC O AgIO (1 mol%) OH H O 3 O A HN B TBHP N R' + R' R A N B H R NH2 N O H O O H J. Am. Chem. Soc. 2006, 128, 13064 oxazetidine OH O 1) 10 - 15 mM OH O 40-60 oC O A B H NHBoc N O NIS, base Ar O 2) pH = 10.5 N Br H O H A N B + H N OMe H O, 0 oC O (O to N acyl H Ar 2 2 N OMe O N BocHN N shift) NO2 H H oxaproline Me O O Me O Nature 2010, 465, 1027 BF OEt , DCM MeO O 3• 2 MeO O o o O CN O -40 C to 0 C O Me O NH N OH N OH O Fmoc BocHN BocHN OBn SH + TsOH• HN CHCl3 N OMe OMe O OBn oxazetidine Me O building block J. Am. Chem. Soc. 2010, 132, 4098 Perspectives and Future Directions The State-of-the-art: • Improvements in coupling reagents and protecting groups allows the construction of most amide bonds (particularly between proteinogenic amino acids) • Routine preparation of small to medium peptides by SPPS is generally rapid and efficient • Small proteins and enzymes are now accessible by chemical ligation methods (NCL, Staudinger ligation, KAHA, etc) employing unprotected peptides from SPPS

Some Ideals: • Facile synthesis of peptides bearing non-proteinogenic or heavily modified amino acids • Unnatural amino acids employed in ligation chemistry (oxazetidines, thiol amino acids, ketoacid precursors) should be commercialized and cost-effective (as accessible as coupling reagents) • Rapid and efficient on-resin ligation chemistry: e.g. an automated, programmable solid-phase "ligation machine" for protein synthesis

Unmet Challenges: • "inaccessible" structures: modified peptides (bearing complex post-translational modifications or non-native structural motifs) O H N 13 N H O H Ph Astexin-1 S O H N N 22 H Subtilosin A O S to !-C cross-links 12