Chemical ligation procedures in peptide and protein chemistry Gábor Mező Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös L. University Budapest, Hungary ERASMUS Teaching Mobility Grant Konstanz 30th October 2008 a) Stepwise synthesis: Z-NH-CHR2-CO-X + NH2-CHR1-CO-OY(NH2) - HX Z-NH-CHR2-CO-NH-CHR1-CO-OY Cleavage of amino protecting group NH2-CHR2-CO-NH-CHR1-CO-OY + Z-NH-CHR3-CO-X - HX Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY cleavage coupling repetition till the end of the peptide sequence Z-NH-CHRn-CO----------------NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY final cleavage step NH2-CHRn-CO----------------NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH b) Fragment condensation Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY cleavage of the carboxyl protecting group cleavage of the amino protecting group Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH NH2-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY + coupling agents - H2 O Z-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OY Final cleavage step NH2-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-NH-CHR3-CO-NH-CHR2-CO-NH-CHR1-CO-OH Limitations of stepwise synthesis Consider a 100 amino acid residue synthesis in one run Coupling yield (%) Final yield (%) 99.99 99.00 99.00 36.60 1.4 81mer 13.26 Low yield; VERY complex crude product; Purification problems. AUFS (214nm) 98.00 90mer 64mer 1.2 1 0.8 0.6 0.4 0.2 0 20 22 24 26 28 30 32 34 36 Time (min) Chromatograms from Hubert Gaertner and Matteo Villain Solutions: a) higher coupling efficiency b) capping after each coupling step c) preparation of several smaller segments, followed by their ligation Chemical ligation a set of techniques used for creating long peptide or protein chains Fragment condensation Synthesis of (semi) protected peptides (Convergent solid phase peptide synthesis; CSPPS) Chemical ligation Synthesis of unprotected peptides by conventional SPPS selective coupling in aqueous solution special resins (e.g. 2-ClTrt) different types of bonds solubility problems up to 200-300 amino acid residues difficult purification fragment condensation in organic solvents amide bond formation up to 100-150 amino acid residues P. Lloyd-Williams et al.: Tetrahedron 49, 11065 (1993) Chemical ligation methods Chemoselective ligation Prior thiol capture Native chemical ligation Expressed protein ligation Acyl initiated capture Staudinger ligation other than amide bond amide bond Chemoselective ligation bromo-acetamido O NH NH2 hydrazide NH HN H2NO hydroxyl-amine SH O aldehyde NH2 N-term. cysteine O O N hydrazone aldehyde O aldehyde O O O O O thioether O O NO oxime O S NH thiazolidine O O S NH HN C-term. cysteine Br NH2 O NH O O SH thioester HN NH2 bromo-acetamido HN O S NH O thiocarboxylic acid Br O O SH NH Resulting bond HN O Component B HN Component A Efficient chemical ligation via thioether bond formation S. Futaki et al.: Bioorg. Med. Chem. 5, 1883 (1997) Synthesis of a four-helix-bundle protein: SPPS, Fmoc/tBu, TMSOBr cleavage SH Ad Cys(Ad)- CH3CO-CGG-ELEELLKKLKELLK-GGCY-NH2 Acm BrCH2CO-GG-ELEELLKKLKELLK-GGCY-NH2 linker Cys(Ad)- -Cys(SH) Br- = -Cys(Acm) linker helix Cys(Ad)- -Cys(Acm) -Cys(Acm) -Cys(Acm) 0.1M Tris (pH 8) 100 eq AgOTf/TFA 6M GnHCl RT, 18h 0oC, 1.5h ( then DTT) -Cys(SH) Br- + S Cys(Ad)- -Cys(Acm) S Br- Cys(Ad)- S 100 eq AgOTf/TFA 0oC, 1.5h ( then DTT) S S Br Cys(Acm) S -Cys(Acm) Cys(Ad)- Cys(SH)- -Cys(Acm) S -Cys(SH) S 1. 1M TFMSA-thioanisole/TFA 2. AgOTf/TFA (both on 0oC, 1.5h) S S S = S S S Cys(SH)- S S -Cys(SH) air oxidation 0.1M NH4HCO3 S S cpeptide= 0.05mg/mL 20oC, 16h S S Decomposition of bromoacetylated compound was observed under acidic cleavage or storage in the freezer. (Yield 18% according to this method) Application of chloroacetylated version gave better results (Yield 30%). More stable derivative. But it needs longer reaction time. ClAc-Peptide + KI IAc-Peptide J.A. Wilce et al.: J. Biol. Chem. 276, 25997 (2001) Synthesis of chloroacetylated oligotuftsin (OT20) carrier and its ligation with Ab(4-10)Cys epitope peptide M. Manea et al. (2008) Biopolymers, 90, 94-104. Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(Fmoc)-Gly]4-MBHA Fmoc-cleavage (2% piperidine-2%DBU in DMF) Boc-synthesis Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(H-Gly-Phe-Leu-Gly)-Gly]4-MBHA Chloroacetylation ( 5equiv ClAc-OPcp) Ac-[Thr(Bzl)-Lys(ClZ)-Pro-Lys(ClAc-Gly-Phe-Leu-Gly)-Gly]4-MBHA HF-cleavage (10 mL HF – 0.5g p-cresol – 0.5g p-thiocresol) Ac-[Thr-Lys-Pro-Lys(ClAc-Gly-Phe-Leu-Gly)-Gly]4-NH2 H-Phe-Arg-His-Asp-Ser-Gly-Tyr-Cys-NH2 0.1M Tris buffer (pH 8) (HCl elimination) Ac-[Thr-Lys-Pro-Lys(CH2CO-Gly-Phe-Leu-Gly)-Gly]4-NH2 S H-Phe-Arg-His-Asp-Ser-Gly-Tyr-Cys-NH2 3h 10.2 22.8 Ab4-10C monomer 23.5 0,6 3910.1 Ligation of Ab(4-10)Cys with OT20(ClAc) followed by HPLC and MS 21.2 0,4 25.4 Ab4-10C dimer 14.9 A220 nm 0,5 2000 3000 4000 5000 m/z 4856.8 27.9 0,3 0,2 5 10 15 20 25 30 Time(min) 6749.9 3373.0 2000 3000 3000 4000 5000 6000 7000 4000 5000 6000 5801.8 2000 m/z 2000 3000 4000 5000 6000 7000 m/z m/z Ac-[TKPK(S-CH2CO-GFLG)G]4-NH2 1,0 3842.8 0,9 0,8 7696.5 H-FRHDSGYC-NH2 21.2 24h 0,5 15.0 0,6 10.2 A 220 nm 0,7 0,4 0,3 0,2 10 15 20 25 30 35 3000 900 4000 983.6 983.7 Time(min) 1100 1300 1500 1700 1900 2100 m/z 1100 5000 6000 7000 1964.8 5 1600 2100 2600 m/z 8000 m/z Thioether bond formation by the aid of a bifunctional coupling reagent NH2 O O NH2 + NH2 N O O KLH N MBS N-(3-maleimido-benzoyloxy)succinimide O T. Kitagawa, et al.: J. Biochem. 79, 233 (1976) O PBS-DMF, 30 min, RT Sephadex G25, 10mM PBS (pH 6) H-9LKNleADPNRFRGKDL22C-NH2 NH2 H O O NH2 N O NH O NH2 S O O H-9LKNleADPNRFRGKDL22C-NH2 PBS solution, pH 7.5 NH2 N O NH O Synthesis of peptide dendrimers with oxime, hydrazone, and thiazolidine linkages J Shao, JP Tam JACS 117, 3893 (1995) O O NH2OCH2C- VA20 CHOCO CHOCO Lys Lys-Ala-OH CHOCO Lys CHOCO VA20 -CCH2ON=C-C)4-TLCP (OxmVA20)4-TLCP O O NH2NHC(CH2)2C- VA20 O 1. NaOH O O VA20 -C(CH2)2CNHN=C-C)4-TLCP (HydVA20)4-TLCP O SH NH2CH-C- VA20 O VA20 -C- OO (HC-C)4-TLCP O VA20 = VMEYKARRKRAAIHVMLALA model peptide with helical structure S N O -C)4-TLCP (ThzVA20)4-TLCP 2. HCl (MeO)2CHCO (MeO)2CHCO Lys Boc Lys-Ala-OMe (MeO)2CHCO Lys (MeO)2CHCO 1. TFA 2. (MeO)2CH2COONa+BOP/DIEA (dimethyl acetal form) Boc Lys Lys-Ala-OMe Boc Lys Boc Rate of (OxmVA20)4-TLCP formation reaction conditions (22oC) (37oC) pH: 4.7 4.2 5.2 5.7* 50% of AcN DMF DMSO 52h 64h 38h 32h (1.0) (0.8) (1.4) (1.6) 35h (1.5) 18h (2.9) 8h (6.5) 23h (2.3) 4.5h (12) Rate of (HydVA20)4-TLCP formation reaction conditions (22oC) pH: 4.7 5.2 44h (0.9) (37oC) 5.7* 50% of AcN DMF DMSO 40h 34h (1.0) (1.2) 76h (0.5) 16h (2.5) 2h (20) 26h (1.5) 1.5h (27) Rate of (ThzVA20)4-TLCP formation reaction conditions (22oC) pH: 4.0 30h (0.8) (37oC) 4.5* 5.0 50% of AcN 24h 16h (1.0) (1.5) 18h (1.3) 8h (3) DMF 5h (4.8) 30h (0.8) 2h (12) in case of 2.5 equiv peptide to a coupling site (10 equiv to lysine dendrimer) * This buffer was mixed with the organic solvents. TFE Influence of different chemical linkages and epitope orientations on biological activity W. Zeng et al.: Vaccine 19, 3843 (2001) B-cell epitope: EHWSYGLRPG (LH-RH) T-cell epitope: GALNNRFQIKGVELKS (HA2 of influenza virus hemagglutinin) -NH-CH2-CO(CH2)4 T-B; amide NH2 Tandem linear SPPS -NH-CH2-CONH2 T-S-B; thioether CH2 S-CH2-CO- T-cell (Cys, N- or C-terminus) and B-cell (BrAc, N-terminus) -NH-CH2-CONH2 (CH2)4 T-oxm-B; oxime NH-COCH=NOCH2CO- T-K-B; amide NH2-CH2-COCH2 S-CH2-COB-S-T; thioether CH-CO- NOCH2-COB-oxm-T; oxime T-cell (Ser at N-terminus or at eNH2 of Lys to aldehyde) and B-cell (aminooxyacetyl group at N-terminus) -NH-CH2-CONH2 NH2-CH2-CO- CH2 CH2 S S NH2-CH2-CO- T-S-S-B; disulfide CH2 T-cell (Cys(Pys) either at N- or C-terminus) and B-cell (Cys at N-terminus) S CH2 NH2-CH2-CO- O O H2N H2N OH OH B-S-S-T; disulfide Immunogen NaIO4 HN O NH2 OH HN O S O T-B T-K-B T-S-B B-S-T T-oxm-B B-oxm-T T-S-S-B B-S-S-T Total yield (%) 45 45 47 43 26 23 21 20 Vaccine Average anti-LHRH titres (log10) 2weeks 9 weeks 20 weeks after the second dose Pregnancy T-B 5.28 4.72 4.26 0/5 T-K-B 5.26 4.96 4.66 0/5 T-S-B 5.14 4.90 4.47 0/5 B-S-T 5.08 4.52 4.16 1/5 T-oxm-B 4.32 4.16 3.86 0/5 B-oxm-T 4.74 4.36 4.14 1/5 T-S-S-B 3.66 3.24 3.22 5/5 B-S-S-T 3.74 3.36 3.33 4/5 Saline control 1 1 1 5/5 Disulfide bonds are less stable in vivo than thioether or oxime bonds Stability study of disulfide bond Z. Bánóczi et al.: Bioconjugate Chemistry 18, 130 (2007) Hca-RQIKIWFQQNRRMKWKKC-NH2 S penetratin S Ac-CSK(Flu)PIIGPDDAIDALSSDFTS-NH2 calpastatin (calpain enzyme inhibitor ) COOH O O O HO HO O O 4-(7-hydroxycumaril) acetic acid (Hca) lex = 360 nm lem= 480 nm HO O OH 5(6)-carboxyfluoresceine (Flu) lex = 492 nm lem= 517 nm Flu Hca COS-7 cells after 4h incubation with conjugates: measurement by fluorescence microscopy Highly enantioselective amide ligation by prior thiol capture D.S. Kemp, D.R. Buckler: Tetrahedron Letters 32, 3013 (1991) First ligation procedure that results in amide bond between unprotected peptide fragments. The incorporated template helps the intramolecular acyl transfer. O Cys(Scm)--- + O SH NH3-CH-COCH2 O S S Cys(Acm)--- Scm: methoxycarbonylsulphenyl HCO2H-HOAc-DMF (1:8:1, v/v) OMe Fmoc synthesis on S-S linked dibenzofurane linker Cleavage of the peptide with Bu3P Cleavage of the protecting groups with TFA ScmCl HFIP-water (4:1, v/v) 30 min O O + NH3-CH-COCH2 S S + O NH3-CH-COCH2 O S S DMSO solution + AgNO3 + DIEA O O O-N acyltransfer 2h, RT OH NH2-CH-COCH2 S S O NH-CH-COCH2 S S Et3P dioxan-water O NH-CH-COCH2 HS Yield of the ligation step is ca. 80% Native chemical ligation (thioester based orthogonal ligation) P.E. Dawson et al.: Science 266, 776 (1994) The amide bond is formed without the aid of a template (difference from the „prior thiol capture“ procedure) N-terminal fragment in thioester form, C-terminal fragment with Cys at its N-terminus + O SR NH3-CH-COCH2 -S C-terminal amino acid should not be Val, Ile, Thr (branching on b-C atom) R good leaving group e.g. red. Ellman`s reagent 5-thio-2-nitrobenzoic acid or thiophenol (thiol ester exchange) in water at pH 7 Chemoselective reaction (reversible) Other Cys without H N 2 any protection CH-COO S CH2 Spontaneous rearrangment S,N-acyl transfer (irreversible) Available for Cys-containing peptides O or de novo peptides with an incorporated NH-CH-COCys, that can be used for another ligation CH2 step HS Native chemical ligation resulting in a methionine in the sequence J.P. Tam and Q. Yu: Biopolymers 46, 319 (1998) + O NH3-CH-CO(CH2)2 -S SR Hcy instead of Cys Chemoselective reaction (reversible) H2N O S Spontaneous rearrangment S-N acyl transfer (irreversible) O NH-CH-CO- (CH2)2 HS CH-CO(CH2)2 methyl p-nitrobenzensulphonate Selective methylation O No other Cys should be in the sequence ! in water at pH 7 NH-CH-CO(CH2)2 CH3S Met Native chemical ligation with Asp or Glu as C-terminal residues M. Villain et al.: Eur. J. Org. Chem. 3267 (2003) After native chemical ligation 20-30% isomer (b- (Asp), g (Glu) peptide bond) Migration of thioester moiety The side chain of Asp or Glu must be protected Boc-chemistry is prefered for the synthesis of thioester Side chain protecting groups must be stable under acidic conditions Clevable by base or reduction (hydrogenation) In case of Glu: Cleavage of BrZ from Tyr OFm alkylation can occur! O -CO-O O S O-CO9-fluorenylmethyl ester (OFm) 20% piperidine-20% DMF-10% 2-mercaptoethanol (pH 13) for 15 min, RT (phenylsulphonyl)ethyl ester 0.1M Na2CO3-10% 2-mercaptoethanol (pH 9) for 2h, 37oC Alkaline solutions can not be used in case of Asp(X) containing peptides because of aspartimide formation: H 2C NH CH O O C C OX C CH2-SH NH CH O C H2C - XOH NH CH2-SH N CH CH C O C O O -Asp-Cys- -Asu-Cys- +H2O +H2O O H 2C NH CH C C CH2-SH O OH CH2-SH NH CH O ~30% a-Asp-peptide C O H2C NH C N CH C O CH C O OH ~70% b-Asp-peptide In case of Asp: CH3 -CO-O O Stability of esters under ligation conditions phenacyl ester (OPac) 2,2,2-trichloroethyl ester (OTce) (2“,4“-dimethoxy)phenacyl ester (OdiMePac) 1-methyl-2-oxo-2-phenyethyl ester (OMop) 1.2 h 1.5 h 3.8 h 9.9 h 1-methyl-2-oxo-2-phenylethyl ester Zn/30% AcOH reduction for 30 min, RT Synthesis: Boc-strategy, on MPAL (b-mercaptopropionic acid-Leu) modified resin in situ neutralization (sensitive thioester bond), HBTU activation. Boc-Asp(OMop)-OH were activated by PyBOP and attached to SH-group. HBTU: O-(Benzotriazol-1-yl)-N,N,N´N´-tetramethyluronium-hexafluorophosphat PyBOP: (Benzotryazol-1-yloxy)-tripyrrolidinophosphonium-hexafluorophosphat Ligation: 1 equiv N-terminal and 1.5 equiv C-terminal fragments dissolved in 0.2 M phosphate buffer containing 6 M Gn.HCl (pH 7.5); Add 1% thiophenol (catalyst) and 1% benzylmercaptan, then adjust the pH to 6.5; ligation is ready in 1h at RT. H-LYRAD(OMop)-SR + H-CSYRFL-OH H-LYRAD(OMop)CSRFL-OH H-LYRADCSRFL-OH Experiments with Na-(1-phenyl-2-mercaptoethyl) auxiliary group P. Botti et al.: Tetrahedron Letters 42, 1831 (2001) The drawback of the mentioned native chemical ligation is that a Cys is needed in the sequence. New procedure: O Br + HS CH3 H3C O CH3 S H3 C SYRFL- R O HN SYRFL- R O O S O CH3 CH3 S BH3 *THF H3C O R : PAM HN HF SYRFL-OH O H3C CH3 NH2-O-CH3 N O 1-(4-methoxyphenyl)-2-(4´-methylbenzylthio)ethylamine Br CH3 S DMF H3C O NH2 H3C O DIEA O SH Na-(1-(4-methoxyphenyl)-2-mercaptoethyl-peptide PEPTIDE -COSR HN ligation PEPTIDE –CO-N O SH SYRFL-OH O SH R O SYRFL-OH CH 3 R O CH3 HF (R = H) or TFA (R = OMe) PEPTIDE –CO-NH-GSYRFL-OH PEPTIDE –COSR Phe-Gly-Gly Phe-Gly-Gly TBRA 1-67 (His) Mouse Larc 1-31 (Ala) N-auxiliary I II II I II MCP1 1-35 (Lys) I reaction time ligation yield removal 16 16 16 16 40 16 40 16 40 >98 >98 87 81 92 73 85 69 76 HF TFA TFA HF HF TFA TFA TFA-TMSBr TFA-TMSBr Ligation: 6M Gn.HCl in phosphate buffer (pH 7), 25oC 1.5 equiv thioester part, 1 equiv N-auxiliary part Native Chemical Ligation at DBSB size synthetized failed 41-70 45 1 71-100 37 5 101-130 24 5 131-164 9 1 unpublished data from Hubert Gaertner and Paolo Botti Expressed protein ligation Native chemical ligation: from unprotected linear peptides Can be prepared by recombinant-DNA-based expression methods Preparation of a C-terminal fragment with Cys at its N-terminus Production of a thioester of the N-terminal fragment 1. Express in E. coli ; 2. Affinity purification RECOMBINANT POLYPEPTIDE -CO-NH-Cys- INTEIN CBD Chitin beads CBD Chitin beads HS NH2-Cys- INTEIN RECOMBINANT POLYPEPTIDE –CO- S transthioesterification HSR RECOMBINANT POLYPEPTIDE –CO-SR Acyl initiated capture Similar to native chemical ligation, but not from a thioester derivative R SH O thioacid R Br + H2N O Br-Ala alkaline conditions S OH N 2 O S-N acyl shift HS C SH Thiol trityl resin Substitution at the highly hindered trityl thiol can be very difficult. Long reaction time and highly activated reagents may be required for acylation. R HN O O Cys containing peptide Bidirectional tandem pseudoproline ligation Z. Miao, J.P. Tam: JACS 122, 4253 (2000) Available for the synthesis of Pro rich peptides and proteins H O O O HX + H2N HX H R O R X N O O X = S and R = H (S Pro) X = O and R = H (O Pro) X = O and R = CH3 (O ProMe) O O O X = S and R = H (Cys) X = O and R = H (Ser) X = O and R = CH3 (Thr) HO N R O,N-acyl transfer Ring chain tautomerization H X O R N H O O Thiaproline formation runs in aqueous solution Oxaproline formation runs in anhydrous pyridine- AcOH H O Ser/Thr- Cys- + O O 1. 2. Ser/ThrO pH 5.3, 10 h, RT thiazolidine formation pH 6.6, 20 h, RT O,N-acyl shift Yield 86% -S ProH O Yield 78% Pyr/AcOH (1:1, v/v), 35 h, RT O -O Pro- -S Pro- Peptide fragments: Cys-Phe-Lys-Ile (C-terminal fragment) Ser-Leu-Ile-Leu-Asn-Gly (middle fragment) Asp-Ser-Phe-Gly (N-terminal fragment) Final product: Synthesis on normal (PAM) resin Synthesis on cyclic acetal resin Asp-Ser-Phe-Gly-O Pro-Leu-Ile-Leu-Asn-Gly-S Pro-Phe-Lys-Ile Synthesis of Bac 7 antimicrobial peptide (59 amino acids) from fragments 1-24, 25-38, 39-59 by the same procedure resulted in peptide(s) that has a little change in the secondary structure (determined by CD) and biological activity. HO O O Staudinger ligation H. Staudinger, J. Meyer: Helv. Chim. Acta 2, 635 (1919) P: + N=N-N triphenylphosphane + - azide P N P+ N- iminophosphorane aza-ylide - N2 P =N-N=N N P N phosphazide N Reactions of aza-ylide H2O H2N-R´ -R3PO O R´´ R R-P-N + R R´ -R3PO R´´ H R´´ O NH-R´´´ -R3PO R´´-N=C=O -R3PO R´´ N R´ H Less reactive carbonyl electrophiles (e.g. amides, esters) can undergo this reaction, especially if the electrophilic attack proceeds in an intramolecular fashion N R´ NH-R´´´ R´´-N=C=N-R´ (phosphane oxide derivative) Advantages of Staudinger ligation: The azide moiety is absent in almost all naturally occurring compounds („bioorthogonal“) Despite their high intrinsic reactivity, azides undergo a selective ligation with a very limited number of reaction partners The azide group is small and can be introduced into biological samples without altering the molecular size significantly M. Köhn and R. Breinbauer: Bioorganic Chemistry 43, 3106 (2004) minireview Staudinger ligation as peptide ligation procedure B.L. Nielsen et al.: Org. Lett. 3, 9 (2001) O H3C N H HS-CH2-PPh2 DCC/HOBt OH S O O N3 R + + PPh2 PPh2 N H O amidophosphonium salt - HS O H N O R N H Ph Other than Gly Much lower efficiency Ph N-peptide2 peptide1 O Ph N H H3C - N-peptide2 H2O PPh2 THF/H2O (3:1) RT, 12 h 90-99% O iminophosphorane -S S O PPh2 N3-peptide2 - N2 S N H phosphinothioester O peptide1 H3C in DMF, RT 95% N-acetyl-glycine peptide1 O P=O Ph NH-peptide2 peptide1 O Presence of Lys (eNH2-group) might cause problems Preparation of azido derivatives from amino acids by diazo transfer J. Zaloom and D. Roberts: J. Org. Chem. 46, 5173 (1981) O H2N CH C X R + CF3SO2N=N=N trifluormethanesulfonyl azide (triflyl azide) Explosive! X = OH, OR´, NHR´ - + N=N=N O CH C X + CF3SO2-NH2 triflamide R Azido-amino acid derivative Amino acid is dissolved in 1N NaOH (pH 9.5) Add 1 equiv triflyl azide dissolved in DCM (~ 1M solution) Stirring overnight (keep the pH between 9-9.5) Phase separation Add 1 equiv of 1M aqueous HgOAc to the aqueous phase Filter off the mercuric salt of triflamide The pH is adjusted to 3.5 with 2N H2SO4 then evaporation The product can be solidified by adding DCHA (salt formation) Leu Phe Glu Met Trp Asn Gly-Phe Tyr-Gly Leu-OEt Ala-OEt Phe-OEt 67% 23% 24% 31% 24% 19% 37% 33% 40% 62% 44% Azido-amino acids are stable under alkaline conditions but sensitive in aqueous acidic conditions especially under pH 2. Azido-peptides can undergo racemization in alkaline solutions (e.g. Ala-Phe resulted in 30 % epimerization in 1N NaOH at RT for 5 min). Synthesis of RNAse A, a 124 amino acids containing protein B.L. Nielsen et al.: JACS 125, 5268 (2003) SPPS RNAse A (110-111) cleavage RNAse A (110-111) Kenner-type safety catch resin SPPS N3 S-CH2-PPh2 RNAse A (110-124) RNAse A (112-124) cleavage PEGA resin mRNA translation RNAse A (1-109) S-R + RNAse A (110-124) Cys 61% RNAse A (1-124) RNAse A (110-111) = Fmoc-Cys(Trt)-Glu(OtBu)- RNAse A = N CH CO-Asn(Trt)-Pro-Tyr(tBu)-Val-Pro-Val-His(Trt)-Phe-Asp(OtBu)-Ala-Ser(tBu)-Val3 2 (112-124) RNAse A (110-124) = Fmoc-Cys(Trt)-Glu(OtBu)-Gly-Asn(Trt)-Pro-Tyr(tBu)-Val-Pro-Val-His(Trt)-Phe-Asp(OtBu)-Ala-Ser(tBu)-Val- Kinetically controlled ligation for the convergent chemical synthesis of proteins D. Bang et al.: Angew. Chem. Int. Ed. 45, 1 (2006) Based on the different reactivity of thioesters and on thiazolidine form of N-terminal Cys as a protection. S N H O + S peptide1 HS O H2N peptide2 S R Native chemical ligation S N H O HS HN peptide1 S methoxyamine hydrochloride 2h, RT H2N S R O HS N H O HS peptide1 peptide2 Thiophenol catalyst 0.2M HCl.NH2OCH3 HS O HN peptide1 O peptide2 S R HN O peptide2 S The synthesis can be further carried out either at N- or C-terminus, either with (Thz)-peptide thio-phenylester or Cyspeptide (thioester).