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Amino Acids

, Volume 46, Issue 12, pp 2733–2744 | Cite as

Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin

  • Vivian Asante
  • Jérémie Mortier
  • Gerhard Wolber
  • Beate KokschEmail author
Original Article

Abstract

Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 3.4.21.1) or the aspartic protease, pepsin (EC 3.4.23.1). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.

Keywords

Chymotrypsin Pepsin Aminobutyric acid Difluoroethylglycine Trifluoroethylglycine 

Abbreviations

Abz

o-Aminobenzoic acid

HPLC

High performance liquid chromatography

Fmoc

Fluorenylmethoxy carbonyl

MD

Molecular dynamics

DIC

Diisopropylcarbodiimide

HOBT

1-Hydroxybenzotriazole

HOAT

1-Hydroxy-7-azabenzotriazole

TFA

Trifluoroacetic acid

TIS

Triisopropylsilane

SPPS

Solid phase peptide synthesis

Notes

Acknowledgments

We are grateful to the Rosa-Luxemburg-Stiftung (RLS) and the Deutsche Forschungsgemeinschaft (Research Training Group “Fluorine as Key Element”) for financial support of Vivian Asante. Jérémie Mortier is grateful to WBI—Wallonie-Bruxelles-International for the award of a postdoctoral grant. We also express our sincere thanks to Dr. Allison Berger for her suggestions and careful editing of the text.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1819_MOESM1_ESM.docx (140 kb)
Supplementary material 1 (DOCX 139 kb)

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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Vivian Asante
    • 1
  • Jérémie Mortier
    • 2
  • Gerhard Wolber
    • 2
  • Beate Koksch
    • 1
    Email author
  1. 1.Institute of Chemistry and Biochemistry, Freie Universität BerlinBerlinGermany
  2. 2.Institute of Pharmacy, Department Pharmaceutical and Medicinal ChemistryFreie Universität BerlinBerlinGermany

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