Racemisation of N-Fmoc phenylglycine under mild microwave-SPPS and conventional stepwise SPPS conditions: attempts to develop strategies for overcoming this

• Published in: Journal of peptide science 2012-05, Vol.18 (5), p.302-311
• Main Authors: Elsawy, Mohamed A., Hewage, Chandralal, Walker, Brian
• Format: Article
• Language: English
• Subjects: (1D) and (2D) NMR; conventional stepwise SPPS; DMTMM; ESI-MS; Fmoc-phenylglycine; Glycine - analogs & derivatives; Glycine - chemistry; Magnetic Resonance Imaging; microwave-SPPS; Microwaves; peptide diastereoisomers; Peptides - chemical synthesis; Peptides - chemistry; racemisation; RP-HPLC; Solid-Phase Synthesis Techniques - methods; Spectrometry, Mass, Electrospray Ionization; super active esters
• ISSN: 1075-2617; 1099-1387
• DOI: 10.1002/psc.2398

We have been engaged in the microwave‐solid phase peptide synthesis (SPPS) synthesis of the phenylglycine (Phg)‐containing pentapeptide H‐Ala‐Val‐Pro‐Phg‐Tyr‐NH2 (1) previously demonstrated to bind to the so‐called BIR3 domain of the anti‐apoptotic protein XIAP. Analysis of the target peptide by a combination of RP‐HPLC, ESI‐MS, and NMR revealed the presence of two diastereoisomers arising out of the racemisation of the Phg residue, with the percentage of the LLLDL component assessed as 49%. We performed the synthesis of peptide (1) using different microwave and conventional stepwise SPPS conditions in attempts to reduce the level of racemisation of the Phg residue and to determine at which part of the synthetic cycle the epimerization had occurred. We determined that racemisation occurred mainly during the Fmoc‐group removal and, to a much lesser extent, during activation/coupling of the Fmoc‐Phg‐OH residue. We were able to obtain the desired peptide with a 71% diastereomeric purity (29% LLLDL as impurity) by utilizing microwave‐assisted SPPS at 50 °C and power 22 Watts, when the triazine‐derived coupling reagent DMTMM‐BF4 was used, together with NMM as an activator base, for the incorporation of this residue and 20% piperidine as an Fmoc‐deprotection base. In contrast, the phenylalanine analogue of the above peptide, H‐Ala‐Val‐Pro‐Phe‐Tyr‐NH2 (2), was always obtained as a single diastereoisomer by using a range of standard coupling and deprotection conditions. Our findings suggest that the racemisation of Fmoc‐Phg‐OH, under both microwave‐SPPS and stepwise conventional SPPS syntheses conditions, is very facile but can be limited through the use of the above stated conditions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. We were involved in the conventional and MW‐SPPS of a phenylglycine (Phg)‐containing pentapeptide that showed Phg racemisation according to RP‐HPLC, ESI‐MS, and NMR data. Various synthetic conditions were attempted to reduce Phg racemisation level and to determine at which part of the synthetic cycle the epimerization had occurred. Finally, we succeeded to substantially limit the degree of Phg racemisation through an MW‐SPPS (50 ºC, 22 Watts), utilizing DMTMM‐BF4 coupling agent with NMM activator base and 20% piperidine as Fmoc‐deprotect base.