Stereospeci®c Synthesis of (2S)-2-Methyl-3-(2 0 ,6 0 -dimethyl-4 0 - hydroxyphenyl)-propionic Acid (Mdp) and its Incorporation Into an Opioid Peptide Yixin Lu, Grazyna Weltrowska, Carole Lemieux, Nga N. Chung and Peter W. Schiller* Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 Received 16 October 2000; accepted 13 November 2000 AbstractÐTo examine the eect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospeci®c synthesis of the tyrosine analogue (2S)-2-methyl-3-(2 0 ,6 0 -dimethyl-4 0 -hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-d-Ala-Gly-Phe-Leu-NH 2 turned out to be a quite potent d opioid antagonist and a somewhat less potent m antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to d and m receptors but may be required for signal transduction. # 2001 Elsevier Science Ltd. All rights reserved. Until recently it has been assumed that the presence of a positively charged nitrogen atom in opioid compounds represents an absolute requirement for their interaction with opioid receptors. 1 The general assumption was that the positive charge on the ligand would engage in an electrostatic interaction with the negatively charged side chain of an Asp residue located in the trans-membrane domain III of opioid receptors. Alternatively, it was suggested that a non-ionic interaction, such as chelation of the quaternary ammonium group by multiple aro- matic residues, may be important for the binding of opioid compounds to the d opioid receptor. 2 Recently, somatostatin-derived cyclic hexapeptide analogues lacking a positive charge were shown to be d opioid antagonists with signi®cant d receptor binding anity (K i d =150±1070 nM). 3 A neutral des-amino analogue of a cyclic b-casomorphin analogue also turned out to be a d antagonist with relatively modest d receptor anity (K i d =109 nM). 3 To further investigate the role of the positive charge on the N-terminal amino group of opioid peptides in the interaction with their receptors, we decided to prepare and pharmacologically characterize an enkephalin ana- logue in which the amino group is replaced with the neutral and almost isosteric methyl group. As parent peptide we chose the potent enkephalin analogue H- Dmt-d-Ala-Gly-Phe-Leu-NH 2 because substitution of Dmt (2 0 ,6 0 -dimethyltyrosine) for Tyr 1 in opioid peptides is known to generally increase d and m receptor binding anity by at least one order of magnitude. 4 The repla- cement of the amino group in this analogue with a methyl group required the development of a stereo- speci®c synthesis of (2S)-2-methyl-3-(2 0 ,6 0 -dimethyl-4 0 - hydroxyphenyl)-propionic acid (Mdp). The stereospeci®c synthesis of (2S)-Mdp is outlined in Scheme 1. Iodination of 3,5-dimethylphenol 1 using a literature procedure 5 aorded 3,5-dimethyl-4-iodophe- nol 2 (79% yield), which was then protected as the Boc derivative (98% yield). Heck coupling of 3 with methyl acrylate 6 gave 4 in 61% yield. The trans con®guration of 4 was established by measurement of the coupling con- stant (16.4 Hz) between the two alkene protons. Sub- sequent catalytic hydrogenation, followed by basic hydrolysis aorded acid 6 in 88% yield. Incorporation of Evans' chiral auxiliary (S)-()-4-benzyl-2-oxazolidi- none was performed in the standard manner 7 to yield 7 (81% yield). Asymmetric methylation 8 then furnished 8 as a single diastereomer 9 in 71% yield after chromato- graphic puri®cation. The chiral oxazolidinone auxiliary and Boc group were then removed to give Mdp (10) 10 in 96% yield. Peptides were prepared by manual solid-phase synth- esis using a p-methylbenzhydrylamine resin and Boc 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(00)00660-0 Bioorganic & Medicinal Chemistry Letters 11 (2001) 323±325 *Corresponding author. Tel.: +1-514-987-5576; fax: +1-514-987- 5513; e-mail: schillp@ircm.qc.ca