Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7-selective peptide – protoxin II Jae H. Park,* Kevin P. Carlin, Gang Wu, Victor I. Ilyin and Donald J. Kyle Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N-methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N-methylmorpholine with 2,4,6-collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage-gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Keywords: Nav1.7; air oxidation; 2,4,6-collidine; electrophysiology; ND7/23; patch clamp; ProTx II; PaTx I; PaTx II; GsMTx II; GrTx I; VsTx II; GsAF I; GsAF II; JzTx V; JzTx XII Introduction Mammalian voltage-gated sodium channels (VGSC) are com- posed of a large pore-forming a-subunit and two auxiliary b- subunits that are presumed to modulate channel activity and functional expression in cell membranes. Each a-subunit has four domains (D1–D4), with each domain containing six trans- membrane segments (S1-S6). Nine different isoforms of the a- subunit have been described (Nav1.1–Nav1.9), with the Nav1.4 isoform being mainly expressed in skeletal muscle and the Nav1.5 isoform being mainly expressed in cardiac tissue. The remaining seven isoforms are expressed in neurons, with Nav1.7, Nav1.8 and Nav1.9 being predominantly expressed in the peripheral nervous system [1]. Nav1.7 channels exhibit slow closed-state inactivation, meaning they can respond to slow membrane depolarization [2]. As a result, Nav1.7 channels may act to amplify small excitatory inputs that are close to the resting potential, thus participating in the spontaneous action potentials in DRG neurons during pathological firing [3]. In addition to this mechanistic evidence for Nav1.7 in pain signaling, an impaired response to inflammatory pain stimuli has been described in mice in which Nav1.7 channels have been knocked out in a subset of peripheral nociceptors [4]. In humans, certain nonsense mutations in the SCN9A gene on chromosome 2q24.3 (that encodes the Nav1.7 a-subunit) have been associated with a phenotype unable to perceive certain noxious stimuli, although other sensory perceptions are normal [5]. Other mutations of the human SCN9A gene cause impairment of normal inactivation of the Nav1.7 channel, causing a persistent painful hereditary condition known as paroxysmal extreme pain disorder. A third pathophysiological condition that is hereditarily linked to mutations in the SCN9A gene is primary erythromelalgia. The mutations underlying this condition cause channels to activate at a lower membrane potential, and clinically this condition manifests as severe burning pain in the extremities [1]. Taken together, these mechanistic and behavioral observations have fueled interest in Nav1.7 channels as an important new drug target for treating various human pain conditions. For the purpose of alleviating pain without adverse side effects that are common in existing therapies, the development of a Nav1.7 channel-selective antagonist may be an attractive alternative approach. One strategy for the design of new molecules that are intended to have inherent target specificity is to initiate structure–activity relationship (SAR) studies on key natural products that are known to already exhibit some degree of the desired target specificity. The goal is to optimize the pharmaceu- tical profile of the molecule in parallel with optimization of the target specificity, while at the same time simplifying the molecu- lar structure. The most Nav1.7 isoform-selective, natural product molecule described to date is protoxin II, making it a candidate for this medicinal chemistry strategy. * Correspondence to: Jae H. Park, Discovery Research, Purdue Pharma LP, 6 Cedar Brook Drive, Cranbury, NJ 08512, USA. E-mail: JaeHyun.Park@Pharma.com Discovery Research, Purdue Pharma LP. 6 Cedar Brook Drive, Cranbury, NJ, 08512, USA Abbreviations used: Boc, tert-butyloxycarbonyl; Fmoc, fluorenylmethyl oxycarbonyl; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle medium; DMF, N,N-dimethylformamide; EGTA, ethylene glycol tetra-acetic acid; GSH, reduced L-glutathione; GSSG, oxidized ()-glutathione; HCTU, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3, 3-tetramethylaminium hexafluorophosphate; HEPES, (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid); NMM, N-methylmorpholine; Pbf, pentamethyl- dihydrobenzofuran-5-sulfonyl; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; Trt, triphenylmethyl; Tris–HCl, Trizma W hydrochloride. J. Pept. Sci. 2012 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 7 January 2012 Revised: 1 February 2012 Accepted: 17 February 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/psc.2407