1. Formation of Fmoc-beta-alanine during Fmoc-protections with Fmoc-OSu
Markus Obkircher, Christian Stähelin, Fritz Dick J Pept Sci. 2008 Jun;14(6):763-6. doi: 10.1002/psc.1001.
During the Fmoc-protection of H-alpha-Me-Val-OH, an unknown side product was found and isolated. The characterization using various analytical methods led unambiguously to the result that Fmoc-beta-Ala-OH was formed during the reaction. The reagent Fmoc-OSu was proven to be the source of Fmoc-beta-Ala-OH, following a mechanism that involved many deprotonation and elimination steps and a Lossen-type rearrangement as key sequence. The impurity Fmoc-beta-Ala-OH was found in a variety of reactions in which Fmoc-OSu was applied, either in the reaction mixture or as a contamination of the crude product. Purification of the Fmoc-amino acid derivatives from this impurity incurred high costs and significant reductions in yield.
2. Fmoc-2-mercaptobenzothiazole, for the introduction of the Fmoc moiety free of side-reactions
Albert Isidro-Llobet, Xavier Just-Baringo, Ariel Ewenson, Mercedes Alvarez, Fernando Albericio Biopolymers. 2007;88(5):733-7. doi: 10.1002/bip.20732.
A double side-reaction, consisting in the formation of Fmoc-beta-Ala-OH and Fmoc-beta-Ala-AA-OH, during the preparation of Fmoc protected amino acids (Fmoc-AA-OH) with Fmoc-OSu is discussed. Furthermore, the new Fmoc-2-MBT reagent is proposed for avoiding these side-reactions as well as the formation of the Fmoc-dipeptides (Fmoc-AA-AA-OH) and even tripeptides, which is another important side-reaction when chloroformates such as Fmoc-Cl is used for the protection of the alpha-amino function of the amino acids.
3. The aspartimide problem in Fmoc-based SPPS. Part I
M Mergler, F Dick, B Sax, P Weiler, T Vorherr J Pept Sci. 2003 Jan;9(1):36-46. doi: 10.1002/psc.430.
A variety of Asp beta-carboxy protecting groups, Hmb backbone protection and a range of Fmoc cleavage protocols have been employed in syntheses of the model hexapeptide H-VKDGYI-OH to investigate the aspartimide problem in more detail. The extent of formation of aspartimide and aspartimide-related by-products was determined by RP-HPLC. This study included three new Fmoc-Asp-OH derivatives: the beta-(4-pyridyl-diphenylmethyl) and beta-(9-phenyl-fluoren-9-yl) esters and also the orthoester Fmoc-beta-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-oct-1-yl)-alanine. 3-Methylpent-3-yl protection of the Asp side chain resulted in significant improvements with respect to aspartimide formation. Complete suppression was achieved using the combination OtBu side chain protection and Hmb backbone protection for the preceding Gly residue.
