ORIGINAL ARTICLE Inﬂuence of Leu 5 conﬁguration on the equilibrium constants of complexes of [Leu]-enkephalin with b-cyclodextrin studied by ﬂuorescence spectroscopy, microcalorimetry and 1 H NMR spectroscopy Justyna Mrozek Æ Emilia Sikorska Æ Agnieszka Skwierawska Æ Joanna Malicka Æ Jerzy Karolczak Æ Bogdan Banecki Æ Wieslaw Wiczk Received: 15 October 2007 / Accepted: 22 May 2008 / Published online: 13 June 2008 Ó Springer Science+Business Media B.V. 2008 Abstract Natural enkephalins and their analogues are very important as potential therapeutic agents (analgetics). In this paper we describe the inﬂuence of Leu chirality of cyclic [Leu]enkephalins on the binding constant with b-cyclodex- trin and spatial and mutual orientation of guest and host molecules. The formation of complexes is enthalpy driven for both cyclic [Leu]enkephalins. Moreover, D-conﬁguration of Leu residue causes an increase of the binding constant of cyclic enkephalin compared to L-analogue. An analysis of 2D NMR spectra reveals that, apart from inclusion complex formed by penetration of cyclodextrin cavity from wider and narrow rims by Trp or Leu residue, a side and/or bottom association complexes are formed. Keywords Enkephalin  Cyclodextrin  Fluorescence spectroscopy  Microcalorimetry  NMR spectroscopy  Inclusion complex  Association complex Introduction The main elements of opioid system are endogenous pep- tides which are natural ligands for opioid receptors (l, d, j). Enkephalins are a part of b-endorphin and interact mainly with d-opioid receptors. Among their neurophysiological functions the most important is perception of pain sensation [1–3]. Natural enkephalins are very ﬂexible molecules with many low energy conformations in solution. One of the methods to decrease the mobility of the peptide chain is cyclization [4–6]. It has been shown that the biological activity of cyclic opioid–peptide analogues depends on mutual orientation and conformational freedom of aromatic pharmacophore group [7–10], which in the case of enkephalins occurs at position 1 and 4. To protect peptide bonds from enzymatic degradation and modify conforma- tional freedom both the peptide chain and side chains of aromatic residues, complexation with cyclodextrin is fre- quently used. The a-, b-, and c-cyclodextrins (CD) are polysaccharides consisting of six to eight D-glucopyranose residues, respectively, linked by a-1,4 glycosidic bonds into a macromolecule. Each cyclodextrin has its own ability to form inclusion complexes with various guest molecules with suitable polarity and dimension because of their spe- cial molecular structure—hydrophobic internal cavity and hydrophilic external surface [11, 12]. Moreover, CDs can recognize not only the size and shape but also the chirality of amino acids and their derivatives [13]. However, mole- cules of many peptides and proteins are too hydrophilic and bulky to penetrate entirely into the CD cavity and the topological constrains of the peptide backbone may inﬂu- ence on the formation of inclusion complexes. Thus, their interaction with CDs could only be local meaning that accessible hydrophobic side chains may form inclusion complexes with CDs [14–18]. To further explain the inﬂu- ence of the side chain on interactions of CD with peptides, in this work we present the results of our studies on the binding process of the cyclic analoques of [Leu]enkephalin with b-cyclodextrin in water solution. Because of the Leu J. Mrozek  E. Sikorska  A. Skwierawska  J. Malicka  W. Wiczk (&) Faculty of Chemistry, University of Gdan ´sk, Sobieskiego 18 80-952, Gdansk, Poland e-mail: ww@chem.univ.gda.pl J. Karolczak Quantum Electronics Laboratory, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland B. Banecki Inter-Collegiate Faculty of Biotechnology, University of Gdan ´sk and Medical University of Gdan ´sk, Kladki 24, 80-822 Gdansk, Poland 123 J Incl Phenom Macrocycl Chem (2008) 62:269–278 DOI 10.1007/s10847-008-9467-8