F A C U L T Y O F L I F E S C I E N C E S U N I V E R S I T Y O F C O P E N H A G E N 5.00 6.00 7.00 8.00 9.00 10.00 11.00 7.5 min 1e 1c 5.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 2 UV_VIS_1 min 1b 5.9 Crude H-FYGGFA-pyroGlu-NH 2 (1c) Impurity from SPPS 4.7 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1 UV_VIS_1 min 2f 6.3 1f 7.1 Purified H-FYGGFA-S(CH 2 ) 2 SO 3 H (1f) Fmoc Solid-Phase Synthesis of C-Terminal Peptide Thioesters via Formation of a Backbone Pyroglutamyl Imide Moiety Entry (#) Base (equiv) PyBrOP (equiv) Reaction conditions 1b (%) 1c (%) 1 DIEA, 10 5 O.N., rt 80 15 2 DIEA, 20 10 O.N., rt 62 30 3 DIEA, 40 20 O.N., rt 56 35 4 DIEA, 20 10 1 x 1h, MW, 60ºC 63 24 5 DIEA, 20 10 2 x 1h, MW, 60ºC 24 70 6 DIEA, 20 10 3 x 1h, MW, 60ºC 10 80 7 DIEA, 20 10 1 x 3h, MW, 60ºC 37 60 8 DIEA, 20 10 1 x 1h, MW, 80ºC <1 50 9 DIEA, 20 10 1 x 2h, HS, 60ºC 37 63 10 DIEA, 20 10 1 x 3h, HS, 60ºC 61 32 11 DIEA, 20 10 2 x 2h, HS, 60ºC 12 78 . Introduction The extensive use of C-terminal peptide thioesters in synthetic protein chemistry, i.e. in native chemical ligation or other chemoselective reactions, has provided the impetus for the search for optimal solid-phase strategies for their synthesis. Fmoc-SPPS has become the preferred synthetic route to peptides, however, peptide thioesters are not directly accessible by Fmoc-SPPS owing to the nucleophilicity of the secondary amine required for Fmoc removal. The development of methods for solid-phase synthesis of peptide thioesters compatible with Fmoc chemistry has therefore been a major challenge over the past decade and several approaches have been reported [1-5]. Methods for reliable synthesis of peptide thioesters with a C- terminal achiral glycine have been established [6]. Each of these methods have overcome some of the difficulties involved in the synthesis of peptide thioesters synthesized by Fmoc- chemistry, however, new more general methods are of interest. A. Pernille Tofteng 1 , Kasper K. Sørensen 1 , Kilian W. Conde-Frieboes 2 , Thomas Hoeg-Jensen 2 and Knud J. Jensen 1 1 Department of Natural Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark, apt@life.ku.dk. 2 Department of Protein and Peptide Chemistry, Novo Nordisk A/S, 2760 Maaloev, Denmark. Tabel 1 Different reaction conditions for the formation of the pyroglutamyl imide peptide 1C. Data is obtained by TFA based release of a small amount of peptide to give 1c which was analyzed by LCMS analysis. MW = microwave heating, HS = heated shaker. Entry (#) Thiol (equiv) Base (equiv) Solvent Reaction conditions 1e* Quan. yields (%) 1e* Purified yields (%) Epimeri- zation (%) 1 RH, 50 PhSNa, 10 DMF 4 x 1h, MW, 60ºC ~35 25 20 2 RH, 50 PhSNa, 10 DIEA, 5 DMF 4 x 1h, MW, 60ºC ~39 25 20 3 RH, 50 PhSNa, 10 DCM 4 x 1h, MW, 60ºC ~5 - N.a. 4 RH, 200 PhSNa, 20 - 3 x 1h, MW, 60ºC ~5 - N.a. 5 RH, 200 DIEA, 20 - 3 x 1h, MW, 60ºC ~5 - N.a. 6 RH, 200 DBU, 5 - 3 x 1h, MW, 60ºC ~50 40 40 7 RH, 200 DBU, 5 - O.N. HS, 40ºC ~50 40 40 8 RH, 50 PhSNa, 10 CH 3 CN O.N. HS, 40ºC ~30 - <1 9 RH, 50 PhSNa, 10 THF O.N. HS, 40ºC ~20 - <2 10 RH, 50 PhSNa, 10 Dioxane O.N. HS, 40ºC <5 - <1 11 RH, 50 PhSNa, 10, crownether CH 3 CN O.N. HS, 40ºC ~50 45 <2 12 RH, 50 PhSNa, 10, crownether CH 3 CN O.N. HS, 50ºC ~50 - <5 13 RH, 50 PhSNa, 2, crownether CH 3 CN O.N. HS, 40ºC ~60 45 <1 Here we describe an unprecedented method for activation of a backbone amide in a peptide by formation of a backbone pyroglutamyl imide, which after displacement by a thiol (thiolysis) provides the peptide thioester (Scheme 1). The synthesis of C- terminal peptide thioesters by this strategy would entail anchoring of a C-terminal glutamic acid residue with a selectively removable side-chain protective group to a solid support. General Fmoc-SPPS protocols were applied for the synthesis of peptides prior to activation. . Pyroglutamyl imide formation . Nucleophilic displacement Scheme 1 The strategy for the synthesis of peptide thioesters through a backbone amide activation. Upper case letters referees to resin-bound peptide while the lower case letters referees to peptides after release from the resin. Figure 2 The two diastereomers 1e and 2e (H-FYGGF-D-A-SR) could not be separated on HPLC thus a thiol exchange with MESNa was performed to give 1f and 2f, respectively. These two diastereomers were separated on HPLC. A) LCMS chromatogram of crude 1e using reaction conditions as #13 (Table 2). B) LCMS chromatogram of purified 1e after MESNa exchange to give 1f. The overlay of 2f illustrates the lack of epimerization. To implement this strategy for the synthesis of thioesters a heptamer peptide, related to the enkephalins, was assembled on- resin to provide Boc-FY(tBu)GGFAE(Ph i Pr)-Rink-TG resin (1A). In effect, this is a double-linker system with a C-terminal Glu(PhiPr) moiety on a Rink-amide linker. The selective protection group, 2-phenyl-iso-propyl-group (PhiPr-group), was removed with 2% TFA to give Boc-FY(tBu)GGFAE-Rink-TG resin (1B) before the pyroglutamyl imide was formed. For this difficult acylation we chose PyBrOP for activation of the carboxylate. Systematic variation of PyBrOP, base and auxiliary nucleophile combined with different temperatures and heating conditions provided optimal conditions (Entries 8 or 11, Table 1). Tabel 2 Nuclephilic displacement with thiols under different reaction conditions to give peptide thioesters in solution. R = -S(CH 2 ) 2 COOEt, Following activation by the pyroGlu imide formation the efficiency of nucleophilic release was tested under varying reaction conditions (Table 2). The resin 1C was treated with different thiol/base mixtures in different solvents at different temperatures which released the peptide into solution as a peptide thioester (1d). Furthermore, the peptide thioester was deprotected to give 1e (Figure 2A) which was quantified on HPLC according to a standard (1a). Early problems with epimerization at the C-terminal stereogenic center was solved by a change in solvent to acetonitrile (Tabel 2 and Figure 2B). However, the thiolate was purely soluble in this solvent but the addition of crownether to the solution solved the problem and increased the yields. A new strategy for the synthesis of peptide thioesters using backbone amide activation has been demonstrated. The method relays on a simple glutamic acid linker system which following activation renders the C-N bond susceptible to thiolysis and provide peptide thioesters. The methods have been thoroughly evaluated in regards to optimal reaction conditions and the issue of epimerization has been solved. . Concluding remarks References Crude H-FYGGFA-S(CH 2 ) 2 COOEt (1e) MESNa, 50 mM pH 7.5 Figure 1 Crude LC-MS chromatogram of the pyroglutamyl imide formation using reaction conditions #11 (Table 1)after TFA based release of peptide. 1b is the linear sequence released from the solid support (H-FYGGFAE-NH 2 ). [*] The yields were calculated from a quantification of 1C. A) B) [1] a) B. J. Backes, A. J. Ellman, J. Org. Chem.1999, 64, 2322-2330; b) R. Ingenito, E. Bianchi, D. Fattori, A. Pessi, J. Am. Chem. Soc. 1999, 121, 11369- 11374. [2] a) X. Li, T. Kawakami, S. Aimoto, Tetrahedron Lett. 1998, 39, 8669-8672 [3] a) S. Futaki, K. Sogawa, J. Maruyama, T. Asahara, M. Niwa, Tetrahedron Lett. 1997, 38, 6237-6240; [4] a) P. Botti, M. Villain, S. Manganiello, H. Gaertner, Org. Lett. 2004, 6, 4861-4 864; b) J. D. Warren, J. S. Miller, S. J. Keding, S. J. Danishefsky, J. Am. Chem. Soc. 2004, 126, 6 579-6578; c) A. P. Tofteng, K. J. Jensen, T. Hoeg- Jensen. Tetrahedron Lett. 2007, 48, 2105-2107; [5] J. B. Blanco-Canosa, P. E. Dawson, Angew. Chem. Int. Ed. 2008, 47, 6851- 6855. [6] J. Brask, F. Albericio, K. J. Jensen, Org. Lett. 2003, 5, 2951-2953.