Elsevier

Bioorganic & Medicinal Chemistry

Volume 19, Issue 23, 1 December 2011, Pages 6977-6981
Bioorganic & Medicinal Chemistry

Effect of 2′,6′-dimethyl-l-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs

https://doi.org/10.1016/j.bmc.2011.10.040Get rights and content

Abstract

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH2 or Tyr-c(Xaa-Phe-d-Pro-Yaa)NH2, respectively, where Xaa and Yaa were l/d Asp or l/d Lys. Further modification of these analogs was achieved by introduction of 2′,6′-dimethyl-l-tyrosine (Dmt) instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin by a microwave-assisted procedure.

Dmt1-substituted analogs displayed high affinity at the μ-opioid receptors, remained intact after incubation with the rat brain homogenate and showed remarkable, long-lasting μ-opioid receptor-mediated antinociceptive activity after central, but not peripheral administration.

Our results demonstrate that cyclization is a promising strategy in the development of new opioid analgesics, but further modifications are necessary to enhance the blood–brain barrier permeability.

Introduction

Cyclization is a well recognized powerful tool in peptide chemistry for generating analogs with improved bioactivity. Cyclic peptides, compared to linear peptides, have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectivity, and improved pharmacodynamic properties.1 Cyclization can be achieved through various bridging bonds between peptide ends and/or side chains.2 Cyclic peptides adopt conformations that are better defined than those of their linear counterparts, which often exist in a conformational equilibrium. Indeed, cyclization reduces the molecular conformational freedom, which is responsible for the contemporary activation of different receptors, increases metabolic stability and may increase lipophilicity, which often improves the blood–brain barrier (BBB) permeability of peptides.

Cyclization has been successfully used in the field of opioid peptides. A great number of cyclic analogs of δ-selective enkephalins and deltorphins3, 4, 5, 6, 7, 8 and κ-selective dynorphins9, 10 have been synthesized over the years through a variety of strategies. The endogenous μ-opioid receptor selective endomorphins are more difficult to cyclize due to their short, only tetrapeptide sequence and the lack of the reactive side-chain groups. Therefore, to obtain cyclic analogs of endomorphins different approaches have been used, such as extension of a peptide chain by an additional amino acid or/and functionalization of amino acid side-chains.

Cardillo et al. synthesized a library of cyclic analogs of endomorphin-1 (EM-1) containing a Gly5 bridge between Tyr1 and Phe4 .11 From this library, the cyclopeptide c[Tyr-d-Pro-d-Trp-Phe-Gly] produced significant antinociception in a visceral pain model.12

Purington et al. synthesized a series of cyclic pentapeptides of a general structure Tyr-c[d-Cys-Xaa-Yaa-Cys]-NH2, cyclized via a disulfide bridge, which displayed μ-opioid receptor agonist and δ-opioid receptor partial agonist/antagonist properties.13

In our earlier papers we have described the synthesis and antinociceptive activity of a series of endomorphin-2 (EM-2) and morphiceptin analogs obtained by cyclization through an amide bond between the side-chain amino and carboxy groups of diamino and dicarboxy amino acids introduced into the peptide chain into positions 2 and 5.14, 15

In this report we describe further modifications of these cyclic peptides, in particular introduction of highly lipophilic unnatural amino acid 2′,6′-dimethyl-l-tyrosine (Dmt) into position 1. Replacement of Tyr in opioid peptides by Dmt paved the way for achieving increased bioactivities. Introduction of methyl groups into the aromatic ring of Tyr influences the conformation of the analogs because of the increased bulkiness of the side-chain. The methyl groups on the aromatic ring of Dmt undoubtedly play a dominant role in the interaction within the opioid binding domain by either direct interaction with hydrophobic side-chains of receptor residues in order to align the critical OH group, or by stabilization of a favored cis-conformer prior to and during binding.16 However, Dmt enhances affinities relative to Tyr cognates for both, μ- and δ-receptors, which results in decreased selectivity of Dmt1 analogs.17, 18, 19 Here we wanted to study the impact of the incorporation of Dmt1 into the sequence of cyclic opioid peptides on their binding affinity and antinociceptive activity.

Section snippets

General

All reagents, unless otherwise stated, were purchased from Sigma–Aldrich (Poznan, Poland). t-Butyloxycarbonyl (Boc)-protected amino acids and p-methylbenzhydrylamine (MBHA) resin were purchased from Bachem AG (Bubendorf, Switzerland).

Analytical and semi-preparative RP-HPLC used were Waters Breeze (Milford, MA, USA) with Vydac C18 column (5 μm, 4.6 × 250 mm) and Vydac C18 column (10 μm, 22 × 250 mm), respectively. Mass spectra of peptides were recorded on Bruker Apex Ultra 7T FT-ICR mass spectrometer

Results and discussion

EM-2 and morphiceptin were used as parent compounds for the design of new cyclic pentapeptides of a general structure: Xaa-c(Yaa-Phe-Phe-Zaa)-NH2 (Xaa = Tyr or Dmt, Yaa = d-Lys or d-Asp and Zaa = Asp or Lys) or Xaa-c(Yaa-Phe-d-Pro-Zaa)-NH2 (Xaa = Tyr or Dmt, Yaa = d-Lys or d-Asp and Zaa = Asp or Lys). Peptides were synthesized using standard Boc/Fmoc orthogonal protection on p-methylbenzhydrylamine (MBHA) resin. Cyclization was carried out between the side-chain amino and carboxy groups of diamino and

Acknowledgments

This work was supported by a grant POLONIUM, a grant from Polish Ministry of Science No. 730/N-POLONIUM/2010/0, a grant from the Medical University of Lodz No. 503/1-156-02/503-01, and a grant from the Centre National de la Recherche Scientifique (CNRS, France). The authors wish to thank Jozef Cieslak for his excellent technical assistance.

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