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Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates

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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.

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Notes

  1. Nomenclature of branched chain polypeptides was used in accordance with the recommended nomenclature of graft polymers. Short codes of the polypeptides are based on one letter abbreviation of amino acids. X is a required amino acid in the side chain; m represents the average number of amino acids in the side chain; i represents average degree of substitution of amino acid X (i≤1) (IUPAC-IUB 1984).

Abbreviations

Boc:

Tert-butyloxycarbonyl protecting group

BSA:

Bovine serum albumin

Cf:

5(6)-Carboxyfluorescein

Cf-OSu:

5(6)-Carboxyfluorescein N-hydroxy succinimide ester

DBU:

1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM:

Dichloromethane

Dde:

1-(4,4-Dimethyl-2,6-dioxacyclohexylidene) ethyl protecting group

DEE:

Diethyl ether

DIC:

N,N′-Diisopropylcarbodiimide

DMF:

Dimethylformamide

EDT:

1,2-Ethanedithiol

Fmoc:

9-Fluorenylmethoxycarbonyl protecting group

HF:

Hydrogen fluoride

HOBt:

1-Hydroxybenzotriazole

MBHA:

4-Methylbenzhydrylamine

MTX:

Methotrexate

TFA:

Trifluoroacetic acid

TMSOTf:

Trimethylsilyl trifluoromethanesulfonate

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Authors and Affiliations

  1. MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary

    Mónika Sebestyén, György Kóczán & Ferenc Hudecz

  2. Department of Organic Chemistry, Eötvös Loránd University, Budapest 112, POB. 32, 1518, Hungary

    Ferenc Hudecz

Authors
  1. Mónika Sebestyén
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  2. György Kóczán
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  3. Ferenc Hudecz
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Correspondence to Ferenc Hudecz.

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The authors declare that they have no conflict of interest.

Ethical approval

The study protocol was approved by the Review Board of the UMCG (METc 2008/186) and was in adherence to the Declaration of Helsinki.

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Handling Editor: J. Bode.

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Sebestyén, M., Kóczán, G. & Hudecz, F. Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates. Amino Acids 48, 2599–2604 (2016). https://doi.org/10.1007/s00726-016-2285-1

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  • DOI: https://doi.org/10.1007/s00726-016-2285-1

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