2',6'-Dimethylphenylalanine (Dmp) Can Mimic the N-Terminal Tyr in Opioid Peptides

Abstract

In opioid peptides, the N-terminal Tyr and the Phe at position 3 or 4 are aromatic residues that are necessary for the opioid activity. Recent structure-activity studies have shown that introduction of the artificial amino acid 2Ј,6Ј-dimethyltyrosine (Dmt) in place of the N-terminal Tyr residue is a promising way to greatly enhance receptor affinity and functional potency. [2][3][4][5][6][7][8][9][10][11][12][13] However the Dmt 1 -substitution generally has resulted in low receptor selectivity due to markedly enhanced affinity toward the concomitant receptor vs. the preferred receptor. 3,6,8,12,13) We have recently demonstrated that 2Ј,6Ј-dimethylphenylalanine (Dmp) is an excellent surrogate for the Phe at position 3 in Tyr-D-Arg-Phe-bAla-NH 2 (YRFB), 14) endomorphin 2, 15) dermorphin, 16) and deltorphin II. 16) We also demonstrated that Dmp can be substituted for the N-terminal Tyr residue in the m receptor-selective agonists YRFB 14) and endomorphin 2 15) without a substantial decrease in m receptor affinity and selectivity. In particular, the Dmp 1 -YRFB had somewhat higher m receptor affinity than the parent peptide, although its in vitro bioactivity was slightly lower.14) These findings are interesting because the Dmp 1 -peptides lack a phenolic hydroxyl group at the N-terminal side chain. More importantly, these Dmp 1 -analogues had high m receptor selectivity. These findings contrast with the effects of Dmt 1 -substitution. In the present study, we report further analogues of the d opioid receptor-selective ligands, deltorphin II (DLT: Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH 2 ) and Leu-enkephalin (ENK: Tyr-Gly-Gly-Phe-Leu), containing Dmp or Dmt at position 1. MATERIALS AND METHODSPeptide Synthesis Peptides were generated by solid phase synthesis using a DIC/HOBt-mediated Fmoc strategy, as previously described.15) Fmoc-Dmp or Boc-Dmt used was prepared by the method reported previously. 13,17) For the synthesis of DLT analogues (1, 2), an N-terminal Dmp residue was incorporated using Fmoc-Dmp racemate.17) Cleavage of the peptides from the resin was carried out by treatment with TFA-phenol (95 : 5) at room temperature for 1 h. The diastereoisomeric peptides (1, 2) were separated using medium-pressure HPLC, as described previously.18) The absolute configuration of the Dmp residue in 1 and 2 was determined by the analysis of the HCl hydrolysate of the peptides using chiral TLC plates (CH 3 CN : MeOH : H 2 Oϭ4 : 1 : 1). 17)Analytical HPLC (YMC-Pack ODS-AM-302 column, 150ϫ 4.6 mm) demonstrated that the purity of all peptides was Ͼ96%. All analogues reported here gave satisfactory FAB-MS and amino acid analytical data (Table 1).Receptor Binding Assay The opioid receptor binding assays were performed as described previously. In Vitro Bioactivity Assay The in vitro biological activities were evaluated using guinea pig ileum (GPI) and mouse vas deferens (MVD) tissues, as detailed previously.15) Isolated longitudinal muscle strips from Hartley strain guinea pig (250-300 g) and vas deferens from ddY strain mice (25-35 g) ...

, -Dimethylphenylalanine : A Useful Aromatic Amino Acid Surrogate for Tyr or Phe Residue in Opioid Peptides

“…In a series of DYN(1-13)-NH 2 analogs, Dmp 1 replacement afforded 27 with greater κ -opioid receptor affinity than that of the parent peptide; Dmp 1 replacement also significantly improved κ -receptor selectivity (IC 50 ratios: 27 , 1( κ )/293( μ )/180( δ ) versus DYN(1-13)-NH 2 , 1( κ )/15.6( μ )/40.1( δ )). These results support our recent finding that Dmp is an effective surrogate for the Tyr 1 residue in opioid peptides [ 49 , 53 , 57 ]. Analog 27 , however, exhibited low GPI potency two orders of magnitude less than DYN(1-13)-NH 2 .…”

“…The usefulness of Dmp 1 substitution for Tyr 1 in the δ -opioid receptor-selective ligands, ENK and DT, and the μ -opioid receptor-selective ligands, EM2 and YRFB, has been investigated [ 49 , 53 , 57 ]. Results of receptor-binding and in vitro assays are shown in Table 6 .…”

Bifunctional [2‘,6‘-Dimethyl-l-tyrosine]endomorphin-2 Analogues Substituted at Position 3 with Alkylated Phenylalanine Derivatives Yield Potent Mixed μ-Agonist/δ-Antagonist and Dual μ-Agonist/δ-Agonist Opioid Ligands

“…[33][34][35] The isolated tissues were suspended in organ baths containing balanced salt solutions in a physiological buffer, pH 7.5. Agonists were tested for the inhibition of electrically evoked contraction and expressed as IC 50 (nM) obtained from the dose-response curves.…”

Rapid Synthesis of Boc-2′,6′-dimethyl-l-tyrosine and Derivatives and Incorporation into Opioid Peptidomimetics

“…2′,6′-Dimethyl- l -phenylalanine (Dmp), analogue 3e , has also been incorporated into the endomorphin scaffold and has been shown to improve binding affinity at MOR and DOR compared to the naturally occurring endomorphins when substituted at the third position . Additionally, phenylalanine and derivatives can sometimes serve as suitable replacements for the N-terminal tyrosine in opioid peptides, while still maintaining biological activity. , To our knowledge, compounds 3c , 3d , and 3f have not been examined as Tyr replacements in opioid ligands. The synthesis of all analogues using the microwave-assisted Negishi coupling proved straightforward.…”