Peptide Ring Closing Metathesis: Minimizing Side Reactions in Arodyn Analogs

Abstract

Kappa (κ) opioid receptor (KOR) ligands, particularly antagonists, have received increasing attention due to their therapeutic potential in stress related conditions [1]. The acetylated dynorphin A (Dyn A) analog arodyn (Ac[Phe,Arg,D-Ala]Dyn A(1-11)-NH2, Figure 1), shows potent KOR antagonism in vitro. Arodyn has high affinity (Ki = 10 nM) and selectivity (Ki ratio (κ/μ/δ) = 1/174/583) for KOR [2]. Clinical development of KOR antagonists has been hampered partly because prototypical small molecule KOR antagonists such as JDTic exhibit long-lasting antagonism, lasting for weeks after a single dose [1]. Conversely, linear peptide KOR antagonists such as arodyn are rapidly metabolized and inactivated. Conformational constraint of the flexible linear peptide by ring closing metathesis (RCM) cyclization was expected to enhance its metabolic stability and potentially stabilize the bioactive conformation. Aromatic residues in arodyn contribute to affinity at KOR [4]; we are exploring RCM cyclization employing Tyr(All) residues which retain the aromatic nature of the N-terminal region. In contrast to high yields observed in RCM using allylglycine in Dyn A analogs [5], a mixture of side products (Figure 2) compromised the RCM product yield of arodyn analogs containing Tyr(All) [4]. Olefin isomerization due to degradation products of the Grubbs catalyst could lead to the observed side products [6]. A model dipeptide, Fmoc-Tyr(All)Tyr(All), was employed to explore reaction conditions to enhance yields of the cyclic RCM product, conditions which were subsequently applied to arodyn analogs. Here, we present the results of different strategies to minimize the side reaction, including examination of reagents reported to suppress isomerization [6,7] as well as microwave heating.