Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract

During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a beta-Ala insertion mutant of the mother peptide. The source of the beta-Ala was identified as contamination of the Fmoc-Ala-OH raw material with Fmoc-beta-Ala-Ala-OH. Further studies also demonstrated the presence of beta-Ala in other Fmoc-amino acids, particularly in Fmoc-Arg(Pbf)-OH. In this case, it was due to the presence of both Fmoc-beta-Ala-OH and Fmoc-beta-Ala-Arg(Pbf)-OH. It is concluded that beta-Ala contamination of Fmoc-amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc-amino acid derivatives. The beta-Ala is often present as Fmoc-beta-Ala-OH and/or as a dipeptide, Fmoc-beta-Ala-amino acid-OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of beta-Ala-related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated beta-Ala contamination as a problem in the manufacture of the drug substance.

Fmoc‐2‐mercaptobenzothiazole, for the introduction of the Fmoc moiety free of side‐reactions

“…This higher amount of b-Ala can be inter- preted from 2, which can undergo polymerization and therefore the corresponding peaks can be more difficult to be identified. Furthermore, this scheme agrees with the fact that no formation of Fmoc-AA-b-Ala-OH takes place, as pointed out by Hlebowicz et al 25 Formation of H-b-Ala-OH or H-b-Ala-OSu can take place either during the preparation of Fmoc-OSu or during the preparation of the protected amino acids. However, the HPLC analysis of commercial Fmoc-OSu has revealed the absence of Fmoc-b-Ala-OH and therefore indicated that the side-reaction does probably take place during the protection of the amino acids.…”

“…Furthermore, the formation of the Su ester of b-Ala, 2 which is susceptible to reacting with the free amino acid to give the dipeptide, is formed when Nu 1 ¼ ¼OSu (pathways a and b). This mechanism may also help to explain the larger content found by Hlebowicz et al 25 of b-Ala by amino acid analysis than that found by HPLC (Fmoc-b-Ala-Arg-OH + Fmoc-b-Ala-OH). This higher amount of b-Ala can be inter- preted from 2, which can undergo polymerization and therefore the corresponding peaks can be more difficult to be identified.…”

“…However, lately the use of Fmoc-OSu has been questioned, because it has been shown that commercial Fmocamino acids prepared with Fmoc-OSu were found to contain Fmoc-b-Ala-OH and Fmoc-b-Ala-AA-OH as contaminants (0.1-0.4%). 25 These levels of impurity become highly problematic when manufacturing drug substances. Herein, a discussion regarding the formation of this double side-reaction is carried out as well as an alternative for the preparation of protected amino acids free of side reactions.…”

CHAPTER 6. Amino Acids and Peptides in Ball Milling

“…Notice that in the case of Fmoc-protection the formation of side-products usually observed in solution (i.e. Fmoc-b-Ala-OH or dipeptides Fmoc-b-Ala-AA-OH via the Lossen rearrangement) 32,33 was avoided under mechanochemical activation. From the point of view of environmental impact, especially in the case of Z-and Fmoc-protection, the E-factors 34 were greatly improved compared to classical syntheses in solution (Scheme 6.8).…”

Oxime Carbonates: Novel Reagents for the Introduction of Fmoc and Alloc Protecting Groups, Free of Side Reactions

“…[13][14][15][16]26,27] Nonetheless, the formation of up to 0.4 % of some amino-acid-based byproducts like Fmoc/Alloc-β-Ala-OH and Fmoc/Alloc-β-Ala-AA-OH can occur when using this reagent for α-amino protection. [28] A detailed discussion of the mechanism of this side reaction has been proposed by Isidro-Llobet et al [25] Azide derivatives, which can be used as solids after isolation from the chloroformates [12,29] or formed in situ before reacting with the amino acid, [30] have also been proposed as an alternative pathway to the N-protection of amino acids, although its explosive nature compromises its use in large-scale synthesis.…”