Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates

Sebestyén, Mónika and Kóczán, György and Hudecz, Ferenc (2016) Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates. Amino Acids, 48. pp. 2599-2604. ISSN 1438-2199

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Abstract

Abstract Methotrexate (MTX) conjugates with poly[Lys(DL-Alam)] based polymeric polypeptides are efficient against Leishmania donovani parasite infection, but the mechanism of the effect is not known yet. We prepared therefore the 5(6)-carboxyfluorescein (Cf) labelled oligopeptide [Cf-K(AaAa)]† and the correspoding MTX-conjugates [Cf-K(MTX-AaAa)] as model compounds for structure-activity experiments. The conjugate aimed to be synthesized with solid-phase methodology on MBHA resin with Boc strategy, using Fmoc-Lys(Boc)-OH. However, various side-reactions were identified. Here we report three problems observed during the synthesis as well as solutions developed by us: a) Unexpected cyclopeptide-formation with the lactone-carboxylic group of the Cf was detected, when Cf was attached to the α-amino group of the Lys residue on solid phase. This was avoided by changing the order of Cf incorporation with using Fmoc/Dde strategy. Alternatively, we have built the peptide with Fmoc strategy on solid phase first and performed the labelling with Cf-OSu subsequently in solution. b) During HF cleavage of the protected conjugates, MTX was demonstrated to form adducts with anisole and p-cresol scavengers, and the TMSOTf cleavage methodology was also found to be inadequate due to the large number of side products formed. We report here that using Fmoc/Dde strategy is an appropriate method to circumvent the cleavage with HF or TMSOTf. c) During the coupling of MTX with oligopeptide, structural and stereo isomers are formed. We have described here the suitable conditions of HPLC separation of these products.

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