Angewandte
Chemie
DOI: 10.1002/anie.200903215
Peptide Rotaxanes
Rotaxane-Based Propeptides: Protection and Enzymatic Release of a
Bioactive Pentapeptide**
Anthony Fernandes, Aurꢀlien Viterisi, Frꢀdꢀric Coutrot, Stꢀphanie Potok, David A. Leigh,*
Vincent Aucagne,* and Sꢀbastien Papot*
Rotaxane and pseudorotaxane architectures are starting to
attract interest as a means of molecularly encapsulating
substrates for potential biological applications.[1–5] ꢀNano-
valvesꢁ that can store and release cargo molecules have been
developed,[1] and the encapsulation of squaraine dyes within
rotaxanes has been shown to enhance dye fluorescence and
stability as well as imparting different cell-localization
propensities on the substrates.[2] A stilbene-based rotaxane
has been used to control the reactivity of a dipeptidic thread
against protease hydrolysis,[3] and functionalized rotaxanes
have also been shown to serve as effective vehicles for the
intracellular delivery[4] of fluorescein-derivatized peptides.
Polyrotaxanes have been studied for their potential as drug-
delivery systems.[5] Herein, we report the synthesis and
properties of a rotaxane in which a macrocycle protects an
extended pentapeptide thread from degradation by different
types of peptidases and the cocktail of enzymes present in
human plasma. The glycosidase-catalyzed cleavage of a
carbohydrate unit in a ꢀstopperꢁ of the rotaxane triggers the
release of the bioactive parent peptide through a self-
immolative mechanism (Scheme 1). The demonstration that
a macrocycle is able to protect an oligopeptide of significant
length from degradation by peptidases, whilst simultaneously
allowing an orthogonal enzyme-activated release mechanism
Scheme 1. Enzyme-triggered release of Met-enkephalin 2 from [2]rotax-
ane propeptide 1.
[*] Dr. A. Fernandes,[+] A. Viterisi,[+] Dr. F. Coutrot, Dr. S. Potok,
Prof. D. A. Leigh
to be built into the structure, is an important step toward the
ultimate goal of a ꢀmolecular sheathꢁ peptide delivery system.
Benzylic amide macrocycles can be templated around
dipeptides in which glycine is the N-terminal amino acid
residue, to form [2]rotaxanes in good yields.[6] Unfortunately,
investigation of their efficacy in protecting peptide-based
threads from enzymatic degradation (a common drawback to
the use of oligopeptides in biological applications[7]) has been
hampered because the yield of the rotaxane-forming reaction
decreases rapidly with increasing peptide length.[6c,8] We
envisaged that a solution might lie in employing a bulky
nitrophenol ester[9] as a stopper for an extendable,[10] shorter,
and more efficiently constructed, peptide rotaxane. We used
this synthetic methodology to target a [2]rotaxane 1 based on
Met-enkephalin 2 (H-Tyr-Gly-Gly-Phe-Met-OH), also known
as opioid growth factor (OGF). Met-enkephalin 2 is a
pentapeptide with a range of bioactivities that include
regulation of nociception and also antitumor activity by
control of cell growth.[11] Release of the free pentapeptide was
designed to occur through cleavage of an enzyme-accessible
monosaccharide on one of the stoppers (Scheme 1) using
E. coli b-galactosidase, an enzyme that has been successfully
School of Chemistry, University of Edinburgh
The King’s Buildings, West Mains Road, Edinburgh EH9 3JJ (UK)
Fax: (+44)131-650-6453
E-mail: david.leigh@ed.ac.uk
Dr. V. Aucagne
Centre de Biophysique Molꢀculaire, UPR 4301 CNRS
Rue Charles Sadron, 45071 Orlꢀans, Cedex 2 (France)
E-mail: aucagne@cnrs-orleans.fr
Dr. A. Fernandes,[+] Dr. S. Papot
Universitꢀ de Poitiers, UMR 6514 Laboratoire de Synthꢁse et
Rꢀactivitꢀ des Substances Naturelles
40 Avenue du Recteur Pineau, 86022 Poitiers (France)
E-mail: sebastien.papot@univ-poitiers.fr
[+] These authors contributed equally to this work.
[**] We thank Prof. Jean-Pierre Gesson (Poitiers) for many useful
discussions and the EPSRC National Mass Spectrometry Service
Centre (Swansea, UK) for accurate mass data. This work was
supported by the Science and Technology Department of the French
Embassy in the United Kingdom, the Scottish Executive and the
Royal Society of Edinburgh. D.A.L. is an EPSRC Senior Research
Fellow and holds a Royal Society-Wolfson Research Merit Award.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2009, 48, 6443 –6447
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6443