Poster Presentation 10th Australian Peptide Conference 2013

Heating and Microwave assisted SPPS of C-terminal acid peptides: The truth behind the yield (#190)

Gilles Subra 1 , Cécile Echalier 1 , Soultan Al-Halifa 1 , Aude Kreiter 2 , karine Puget 2 , Jean Martinez 1 , Muriel Amblard 1 , Pascal Verdie 1 , Luisa Ronga 2 , Pierre Sanchez 1
  1. Amino acids, Peptides and Supported synthesis, IBMM institute of biomolecules Montpellier, Montpellier, 34, France
  2. Genepep, Saint Jean de Vedas, France

Microwave (MW) irradiation is now accepted as an alternative and efficient way of heating, speeding up organic reaction.  Applied to SPPS,  the thermal effect of MW12  enable the synthesis of a 20-mer in few hours.  However, despite correct purity of crude peptides prepared in Fmoc/tBu microwave assisted SPPS3 , surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides on trityl resin. This could be explained by cyclisation/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that it is not the case and that this yield loss was due to high temperature promoted cleavage of the 2-chlorotrityl ester, releasing the growing C-terminal acid peptides in solution through a SN1 hydrolysis during MW-assisted SPPS. This mechanism is simiral to which was observed to explain trityl acetate hydrolysis in solution.4

1107-image%20abstract%20trityl.jpg

Three different peptide models (compds 1 2 and 3) were synthesised with and without heating, examining the yields and the side products. Moreover, a tripeptide model was prepared to perform kinetic studies at different stages of the MW assisted SPPS cycle.

Cmpd #

sequence

MW (g/mol)

Yield (%) (RT)

Yield (%) (70 °C)

1

H-EKRYCS-OH

784.8

 64.6

7.2

2

H-GSNKGAIIGLM-OH

1060.3

95

2.6

3

H-WHWLQLKPGQPMY-OH

1684.0

91

0.5

At last we demosntrated that  is possible to get advantage of the thermal instability of trityl-peptidyl resin to cleave in neutral conditions a fully protected peptide. However, due to long reaction times that could be required (> 48 hours), a careful monitoring of the released peptide has to be set up to ensure that side chain protecting groups are not partially removed.

  1. Bacsa B, Horvati K, Bosze S, Andreae F, Kappe Solid-phase peptide synthesis at elevated temperatures - A comparison of conventional and microwave heating technology. J Pept Sci 14 (8):73-73 2008
  2. Barlos K, Chatzi O, Gatos D, Stavropoulos G 2-Chlorotrityl Chloride Resin - Studies on Anchoring of Fmoc-Amino Acids and Peptide Cleavage. Int J Pept Prot Res 37 (6):513-520 1991
  3. Pedersen SL, Tofteng AP, Malik L, Jensen KJ Microwave heating in solid-phase peptide synthesis. Chem Soc Rev 41 (5):1826-1844 2012
  4. Horn M, Mayr H Stabilities of Trityl-Protected Substrates: The Wide Mechanistic Spectrum of Trityl Ester Hydrolyses. Chem-Eur J 16 (25):7469-7477 2010