Fmoc-Lys(Dde)-OH

Fmoc-Lys(Dde)-OH

Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates.[Pubmed:27357306]

Amino Acids. 2016 Nov;48(11):2599-2604.

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 alpha-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.

Strategies for the synthesis of labeled peptides.[Pubmed:19499054]

J Biomol Tech. 2000 Dec;11(4):155-65.

Labeled peptides synthesized by core facilities are frequently used by researchers for following trafficking of a peptide, for binding studies, to determine substrate specificity, and for receptor cross-linking studies.The membership of the Association of Biomolecular Resource Facilities was asked to participate in a study focusing on synthesis of a biotin-labeled peptide, and it was suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used.This strategy can be used for addition of a variety of labels other than biotin and should prove useful to core facilities. Comparison of the new strategy to other strategies was performed. Biotin labeling has long been assumed to be routine and specific. Despite the assumed routine nature of synthesizing biotinylated peptides, 9 of the 34 samples submitted did not contain any of the correct product. Although synthesis using Fmoc-Lys(Dde)-OH plus biotin generally gave the highest yields, other approaches also yielded a high percentage of the correct product.Therefore, the various strategies are generally comparable. The major advantage of this new approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.