title: Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates.
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) labeled oligopeptide [Cf-K(AaAa)] (a: D-alanine, A: L-alanine) and the corresponding 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: (1) 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 labeling with Cf-OSu subsequently in solution. (2) 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. (3) 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.
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary.
authors.forename: Mónika
authors.initials: M
authors.lastname: Sebestyén
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Kóczán
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary. fhudecz@elte.hu.|Department of Organic Chemistry, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary. fhudecz@elte.hu.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Hudecz
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D008727:Methotrexate / Q000737:chemistry*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D008727:Methotrexate
keywords: Fmoc/Dde strategy; Labeling with carboxyfluorescein; Methotrexate; Oligopeptide conjugates