Lara Malins Baran Group Meeting Highlights in …

1 Lara MalinsBaran Group Meeting04/23/16 Highlights in Peptide and Protein Synthesis"My entire yearning is directed toward the synthesis of the first enzyme. If its preparation falls into my lap with the synthesis of a natural protein material, I consider my mission fulfilled."Emil FischerPeptides: The Greatest HitsPeptides: Greatest Hits Collection(1882-present)Scope of this Meeting : (aka Lara's peptide mixtape) peptides - "Love Question Mark" (2013)..Rugby league player suspended after admitting to the "use and trafficking of peptides "(August 2013) Brief historical perspective on peptide synthesis, outlining challenges and discoveries in the early years (1882-1960s) Development of solid-phase peptide synthesis (SPPS) Advances in coupling reagents and protecting groups Chemoselective ligation chemistry for the synthesis of large peptides and proteins Perspectives and unmet challenges A collection of some personal favorites! peptides in the news: Non-amide bond forming ligation methods ( bioconjugation, see Sella GM, May 2014 - ADCs) Biological expression or semi-synthetic approaches ( expressed protein ligation, unnatural amino acid incorporation - cf.)

2 P. Schultz) Unnatural amino acid /peptide derivatives (!- peptides , peptoids, peptidomimetics) Post-translational modifications (glycoslyation, sulfation, phosphorylation) and the synthesis of post- translationally-modified peptides or proteinsThe Golden Oldies: peptides as Therapeutic Leads: ~100 therapeutic peptides currently on market Octreotide, Goselerin, Leuprolide - top sellers Approval rate for peptides (20%) vs. for small molecules (10%) Anticipated global market of US$ billion by 2020 Octreotide(Novartis)" peptides can assist you during your journey to health and well-being" ( )-EuPA Open Proteomics 2014, 4, 58 HNNHOHNOOHNOHNONHOHNSSONH2 NHNH2 OHHOOHONHOOHHNOONHONHNONH2 HNNHNHOHONHOHNONHOHNNHNHNONHONH2 Goselerin(AstraZeneca)Topics not covered:18821901193219531963present19701 9921994dipeptideGly-GlyCbzoxytocinSPPSFm ocNCLligationTimeline:Theodor CurtiusH2 NOAgO+PhClOPhNHOHNOOHOPhOHO++2 AgCl22 PhNHON3OH2 NHNOOEtONHHNOOEtOOHNOPhNHHNON3 OOHNOPh1) H2 NNH22) HNO2 AANHHNONHOOHNOPhAcyl azide couplingHNOOEtOpentaglycine peptideJ.

3 Prakt. Chemie 1882, 26, 145J. Prakt. Chemie 1904, 70, 57 Theodor Curtius- Emil Fischer, 1905(letter to Adolph von Baeyer)HNNHOO conc. HCl"H3 NHNOOHOClBer. Dtsch. Chem. Ges. 1901, 34, 2868 Ber. Dtsch. Chem. Ges. 1905, 38, 605 NHOClOBrH2 NOOEtNHOOEtOHNOBrNHOOHOHNOH2 Nbase/NH3 Emil FischerAcid chlorides in peptide synthesis and the #-bromoacyl methodglycylglycineleucylglycylglycine"m asked" Leu amineH2 NHNONHONONHOHNONH2 OOHNNH2 OOHNONHOOHH2 NSST otal Synthesis of OxytocinVincent du Vigneaud Head of Biochemistry - Cornell University Medical College Studied the biochemical importance of sulfur-containing compounds (insulin, penicillin, oxytocin, vasopressin) First total chemical synthesis of a bioactive peptide hormone Nobel Prize in Chemistry (1955)J. Am. Chem. Soc. 1953, 75, 4879 (Vigneaud) Prepared using a 3-fragment condensation approach Coupling methods: acid chlorides, anhydrides, pyrophosphite method Purified by counter-current distribution (CCD) No HPLC, mass spec, high resolution NMR!

4 Overall yield <<1%CCD apparatus(invented by Lyman Craig)Practical Limitations: lack of readily cleavable N-terminal protecting groups limited access to enantiopure amino acidsOctadecapeptide - FischerLeu-(Gly)3-Leu-(Gly)3-Leu-(Gly)8- GlyBer. Dtsch. Chem. Ges. 1907, 40, 1754 NHOHNOHNOBrOClLara MalinsBaran Group Meeting04/23/16 Highlights in Peptide and Protein SynthesisNHROPGHNR'OOR*NROOR*OPGHNR'NOOP GHNR'RNOOHPGHNR'RHNOOPGHNR'RRacemization through oxazolone formation (major)OONHROONHROONHRCbz (Z)BocFmoc CarpinoJACS 1957, 79, 4427 McKay and AlbertsonJACS 1957, 79, 4686 Bergmann and ZervasBer. Dtsch. Chem. Ges. 1932, 65, 1192 CarpinoJACS 1970, 92, 5748H2/PdNa/NH3 (l)HBr/AcOHTFAHC lbase (amines)NH3 (l)DeprotectionConditions:Essential protecting groups (urethanes)PhNHOClOA convenient 'ORH2NR'racemizationbenzoylONHOClORconfi gurationallystablePhCbzONHONHR'ORPhH2NR' NHR'OOHOR''OBoc, Fmoc, Cbz (racemization-suppressingurethane Group )NHROHNHNR'OOR''OCN321chain growthOMeOH2 NROHN1 OMeO23 NHROHNR'OOR''O23 OHN-acyl groupracemization-proneH2N1 OMeONHROHNHNR'OOR''O21 OMeO3 CNchain growthXC-to-N:N-to-C:epimerization at C-2 Unidirectional chain growth:OxytocinJ.

5 Am. Chem. Soc. 1959, 81, 5688 (Vigneaud)Ber. Dtsch. Chem. Ges. 1932, 65, 1192 Improved oxytocin synthesis published in 1959: stepwise, p-nitrophenyl ester couplings, C-to-N elongation, 38% overall yieldIIIIIIdu VigneaudRacemization through enolization (minor)NHROPGHNR'OOR*NHROPGHNR'OOR*2 Lara MalinsBaran Group Meeting04/23/16 Highlights in Peptide and Protein SynthesisBruce Merrifield and Solid-Phase Peptide Synthesis (SPPS) Biochemist by training, moved to Rockefeller Institute for Medical Research (1949) Work on the structure of peptide growth factors required the synthesis of a pentapeptide:29 May 1959 - Merrifield records concept of SPPS in his lab book"my overall yield of pentapeptide was 7%, and it took me 11 months. Certainly, an experienced peptide chemist would have done better, but not without considerable effort."OOLinkerNHOOL inkerNHOHOLoad resinDeprotectN-terminusH2 NLinkerNHOHONHOOL inkerOHNL inkerPeptide 1 Repeat: deprotectand couplei) Deprotect (global)ii) Cleave from resinPeptide 1H2 NOOH= solid support (resin)= N-terminal amino protecting Group = side-chain protecting groupsSynthetic peptide productcoupleamino acidSolid-phase peptide synthesis (SPPS)iii) HPLC purifyCommon linkers:RClTrityl linker (R = H)2-Cl trityl linker (R = Cl)ClMerrifield (chloromethyl)Linker=ONH2 OMeOMeRink amide linkerHNPAM linker(4-hydroxymethyl-phenylacetamidome thyl)OOHC ommon linkerClDVBcross-linkMerrifield resin Resin loading: - mmol/gram (number of functionalizable sites) "Swellability": determines appropriate solventsProperties to consider:CbzHNOONHEt31)OONHEt32)OOCbzHN1 ) Cbz removal: 30% HBr in AcOH(2-4 h)ClH2 NONHOHNOOHNHOJ.

6 Am. Chem. Soc. 1963, 85, 2149 (Merrifield)1984 - Awarded the Nobel Prize for SPPSA reviewer's thoughts: the approach is "a chemistry at all, a concept which should be surpressed by the community."OOH2N2) Et3N/DMFCbzHNOHODIC, DMF, rt, 18 h1)2) Ac2O/Et3 Nrt, 2 hOOHNOCbzHNSPPS then NaOH/EtOHSeminal PaperBiopolymers 2008, 90, 175 (Mitchell)Leu-Ala-Gly-Val(8% yield of purified compound)MerrifieldH2 NOOONH2H2 NOOOH2 NOOONH2NH2nnn- polystyrene +1-2% divinylbenzene (DVB) cross-link:- ChemMatrix polyethylene glycol (PEG):more polar resindecreases hydrophobic aggregation(PCT Int Appl 2005, WO 2005012277 A1)J. Am. Chem. Soc. 1966, 88, 5319 For development of solid-phase oligonucleotide synthesis, see:3 NNLara MalinsBaran Group Meeting04/23/16 NHOOOO side-chainbenzyl PGsN-terminal BocOOBoc SPPS (Boc/benzyl)Fmoc SPPS (Fmoc/tBu)NHOOOO side-chain tBuor TrtN-terminal FmocOOpiperidine labileTFA labileTFA labileHF labilePros: Optimizable to 50-60 residues (less aggregation prone) Stable thioester linkagesCons: HF safety hazard Limited FG compatibilityPros: mild and safe (no HF required) spectrophotometric quantification of couplingsCons: More aggregation prone Incompatible with base sensitive substrates ( thioesters)"Assembly of the 124 amino acid residues into the protected, resin-bound straight-chain precursor of RNase required 369 chemical reactions and 11,931 steps of the automated peptide synthesis machine.

7 "J. Am. Chem. Soc. 1969, 91, 501 (Merrifield)Merrifield and his peptidesynthesis machineAutomationNHOORH2 NRNHHN-CO2 Fmoc Spectrophotometric Quantificationpiperidine-fulvene adduct!= 7800 cm-1 (" = 301 nm)fastResinHF labileResinTFA labile"Automated Synthesis of peptides "Science 1965, 150, 178 Synthesis of an Enzyme with Ribonuclease A (RNase) Activity N-terminal Boc protecting groups DCC as the coupling reagent - JACS 1955, 77, 1067 (Sheehan)NNNOHNHHOAtHOBtJ. Am. Chem. Soc. 1993, 115, 4397 (Carpino) - 1367 citationsNOOHOHOXt/EDCHOAt: complete at 22 hHOBt: trace productOMeH2 NOOHNHOO+HNNHOOOMeOconditionsDCC, 24 hHOBt, DCC, 24 hHOAt, DCC, 24 hHOAt, DCC, DCM, 24 hDL-isomer (%)coupling < 1-2%HOAt as a peptide coupling additive superior leaving Group neighboring Group effectOONNNXvia:NHRRNNOHNNNNOORI mprovements in Coupling ReagentsHOBt in peptide synthesisChem. Ber. 1970, 103, 788 (K nig) - 965 citationsNNNOHHOBtRacemizationsuppressor Reviews:Chem.

8 Rev. 2011, 111, 6557 (Albericio); Tetrahedron 2004, 60, 2447 (Han)Tetrahedron 1991, 47, 259 (Coste)HOAt:Oxyma: A Safer AlternativeCOOEtNCNOH Slightly more reactive than HOAt Non-explosive (unlike HOBt, HOAt)Chem. Eur. J. 2009, 15, 9394 (Albericio)COOEtNCNOKK-Oxyma: for highly acid -labile resinsEur. J. Org. Chem. 2013, 6372 (Albericio) non-acidic alternative to Oxyma**Final product had enzymatic activity (though less than native RNase)NNNOPNMe2Me2 NNMe2PF6 BOPNNNOPNNNPyBOPPF6 BrPNNNPyBrOPPF6 Tet. Lett. 1975, 16, 1219(Castro) Highlights in Peptide and Protein Synthesis4 NHNHNNH2 OHONHNHNH2 ClH3NH3 NNHNHNH2 ClH3 NNNHHNNH2 OHNEDC/HOBtHATU, HBTU, TCTU, DPPA, T3P, EDC, DIC, DCC, BOP, BOP-Cl, PyAOP, PyBOP, PyBrOP, cyanuric chlorideOther attempts:"Alphabet Soup": Why are there so many coupling reagents?J. Am. Chem. Soc. 2011, 133, 14710 Lara MalinsBaran Group Meeting04/23/16 Coupling of bulky (and/or non-proteinogenic) amino acids Considerations for removal of coupling reagent byproducts Enhancing rate of amide bond formation Minimizing racemization of activated acidCbzHNOHORNNR2 CbzHNOORHNNR2 CbzHNHNORNR2 OCbzHNHNORNR2 OAib couplings with the Aziridine methodHelv.

9 Chim. Acta 1990, 73, 13 Helv. Chim. Acta 1987, 70, 102 (Heimgartner)Et2O, rt, hJ. Org. Chem. 1992, 57, 5566 (Heathcock)Synthesis of (-)-Mirabazole C - Application of PyBroPCbzHNOHOMeBnSH3 NOMeOMeBnSClHNOMeOMeBnSCbzHNOMeBnSPyBroP , DIEADMAP, DCM, 4 h90%1) HBr, HOAcHNOMeOMeBnSNHOMeBnSOCbzHNMeBnSA2) A, PyBroPDIEA, DMAPDCM, 24 hHNNHOMeBnSNHOMeBnSOHNMeBnSOSBnSNNSSNMeM eSNMeSNNSSNMeMeSNMe1) Na, NH32) NH4Cl3) TiCl4, DCMNiO2, benzenereflux, 6 h60%63%+60%(-)-Mirabazole CCase Studies: acid chloride, BOP, BOP-Cl, DPPA, DCC were unsuccessful in coupling A!-aminoisubutyric acid (Aib)H2 NOOHHNOOHMeN-Me-valineSynthesis of Palau'amineNNONNOONNBocHBocOHOHNBocNHNHO HMeO2 CMeBocNNBocHBocFOHS ynthesis of 5-N-acetylardeeminDCC/DMAPDCC/HOBtBOP-Cl C8C15bpartial racemization(C15b or C8)cyanuric fluoridepyridineDCM, -15 oCH2 NCO2 MeMe+H-Ala-CO2 MeNaHCO3, H2 ODCM71% (2 steps)J. Am. Chem. Soc. 1999, 121, 11953 (Danishefsky)See also: TFFH as a reagent for acid -fluoride couplings: JACS 1995, 117, 5401 (Carpino)NNFPF6 TFFHC hoosing a coupling reagent: where to begin?

10 Solid-phasesolution-phaseaniline?acidchl oridebase sensitive?DIC/HOBtDIC/oxymabulky AA,difficultcouplingPyAOPHATUHOAt/DICPyB rOPPyBOPHBTUHOBt/DICgeneralcondsbulky AA,difficultcouplingHOBt/DIC or DCCgeneralcondsHOAt/DICacid fluorideor EDC (water-soluble) Highlights in Peptide and Protein SynthesisStart5 Lara MalinsBaran Group Meeting04/23/16A Few Notable protecting GroupsReview: Chem. Rev. 2009, 109, 2455 (Albericio)SO2 OOBsmocJACS 1997, 119, 9915 JOC 1999, 64, 4324SO2OO!-NsmocSO2OO"-Nsmoc>>JOC 2007, 72, 1729 HJOC 2007, 72, 1729 Relative sensitivity to piperidine:5 g Fmoc-Phe-OH: $ g Bsmoc-Phe-OH: $ comparison (Alfa Aesar)SO2 OOHNClNHSO2CH2H2 NCl-CO2 NSO2NJ. Am. Chem. Soc. 1997, 119, 9915 (Carpino)Leu-Leu-LeuOOONHOSO2NH2 NNH2NH2 HNBsmocMeBsmoc removalonlyFm removalonly10% in DCM2% in DCMA lternatives to FmocMichaeladditionSelective removal of Bsmoc or Fm/FmocJOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.)OOONHOONHOOSO2 NNHOOONHOOH2 NNNO2 SOOHPEGEDC, DMAPDCM, rt, 12 hOOONHOONH82% (2 steps)(1 equiv.)