Proceedings book download - 5Z.com

Table 1. Yields of selected crude peptide thioestersEntry Peptide thioester Yield a (%)1 Ac-SYRGF-S(CH 2 ) 2 COOMe (13a) 69/89 b2 Ac-SYRGW-S(CH 2 ) 2 COOMe (13b) 78 b3 Ac-SYRGQ-S(CH 2 ) 2 COOMe (13c) 864 Ac-SYRPV-S(CH 2 ) 2 COOMe (13d) 675 Ac-SYRGF-MMP (14a) 54/83 b6 Ac-SYRGW-MMP (14b) 90 b7 Ac-SYRGQ-MMP (14c) 538 Ac-SYRGF-MMP (14d) 88a The yields were determined based on weight from the resulting peptide based on the firstamino acid coupling. Purities of products were determined via HPLC at 220 nm withUV/Vis spectroscopy to be >90 % pure. b This peptide yield was attained with the couplingof a dipeptide to the initial amino acid.For investigating the stability of this linker on trityl resin, acetyl-protected 4-mercapto 4-methylpentanol (MMP) 3 was designed as a linker precursor, and model compound 6a wassynthesized (Figure 1). Thioester 6a possessed a half-life of more than 9 days in thepresence of 20% piperidine in DMF. In contrast to the Boc-Phe thioester 6a, thecorresponding thioester of Boc-glycine 6b was synthesized and treated with 20% piperidinein DMF. The stability of 6b in the presence of piperidine was reduced in comparison with6a displaying a half-life of approximately 7.5 hours.Optimized acylation of the thiolinker resin 8 was accomplished with double couplingsof Fmoc-amino acid fluorides delivering yields of 9 of up to 90% under racemization-freeconditions. Diketopiperazine (DKP) formation after the second deblocking step led to onlymoderate loss of yield depending on the peptide sequence. This could be fully avoided bycoupling of dipeptides to the first resin bound amino acid using HATU/HOAt.For a detailed validation of the linker concept and the sequence-dependent comparisonof reactions, a number of N-acetylated pentapeptides of generic structure Ac-SYRX bb X aa11 were synthesized and cleaved off the resin using various conditions. Nucleophiliccleavage gave protected peptides 12a-d and subsequent treatment with TFA furnishedpeptidyl thioesters 13a-d in high purity and excellent yields (Table 1). Acidic cleavage ofresin-bound thioester peptides 11a-d with TFA furnished 14a-d also in high purity andgood yields (Table 1). No cleavage of the MMP-linker was observed in any of theseproducts, no truncated products or failed sequences were detected. NMR analysis ofthioester 14a showed conservation of the thioester carbonyl after TFA cleavage and thusexcluded an S O transesterification of the peptide C-terminus.We have presented, to our knowledge, the first direct synthesis of peptide thioestersusing common Fmoc-based methodology. The MMP-linker can be obtained quickly fromwell accessible starting materials and can be applied for the preparation of protected andunprotected peptide thioesters displaying a broad range of sequences. The products can bedirectly used in native chemical ligations and possibly in other reactions of peptidylthioesters.AcknowledgmentsWe thank André Horatscheck and Dr. Peter Schmieder for technical support, and Dr. ValentinWittmann for helpful discussions. We gratefully acknowledge the DFG (RA895/2, FOR 806 and SFB765) and the Fonds der Chemischen Industrie for continuous support.References1. a) Wieland, T., Bokelmann, E., Bauer, L., Lang, H.U., Lau, H. Justus Liebigs Ann. Chem. 583, 129-149 (1953); b) Dawson, P.E., Muir, T.W., Clark-Lewis, I., Kent, S.B. Science 266, 776-779 (1994);c) Raz, R. Rademann, J. submitted for publication.2. Morse, B.K., Tarbell, D.S. J. Am. Chem. Soc. 74, 416-419 (1952).3. Mohrig, J.R., et al. J. Org. Chem. 72, 793-798 (2007); c) Wallace, O.B., Springer, D.M.Tetrahedron Lett. 39, 2693-2694 (1998).209