*3.7. Solid-Phase Total Synthesis of 1a, 1b*

Compounds **1a**, **1b** were synthesized by 2-chlorotrityl chloride resin (loading 1 mmol/g) as described in Scheme 1. Then, 4 equiv of Fmoc-*D*-Phe (2 mmol) was added to a suspension of 1 equiv of 2-chlorotrityl chloride resin (0.50 g, 0.5 mmol), 4 equiv of HCTU (2 mmol), 8 equiv of DIEA (4 mmol), and DCM/DMF (*v*:*v* = 1:1). After stirring for 2 h, the resin was filtered and washed with DCM/DMF (1:1) for three times. The reaction mixture was treated with piperidine:DMF (*v*:*v* = 1:4) for stirring 30 min to deprotect the Fmoc group. After filtering and washing, the resin was used as resin-*D*-Phe4-NH2 for the next coupling reaction.

A solution of HCTU (2 mmol) and DIEA (4 mmol) in DCM/DMF (1:1) was added to a mixture of resin-*D*-Phe4-NH2, Fmoc-*L*-Val/ Fmoc-*D*-Val (2 mmol). After the reaction mixture was stirred for 60 min, the resin was filtered and washed. To remove the Fmoc group, the same procedure was repeated as above. The resin was used as resin-*D*-Phe4-*L*-Val3-NH2/*D*-Phe4-*D*-Val3-NH2 for the subsequent next coupling reaction, respectively. A solution of HCTU (2 mmol) and DIEA (4 mmol) in DCM/DMF (1:1) was added to a mixture of resin-*D*-Phe4-*L*/*D*-Val3-NH2, Fmoc-*D*-Val/ Fmoc-*L*-Val (2 mmol). After the reaction mixture was stirred for 60 min, the resin was filtered and washed. To remove the Fmoc group, the same procedure was repeated as above. The resin was used as resin-*D*-Phe4-*L*-Val3-*D*-Val2-NH2/*D*-Phe4-*D*-Val3-*L*-Val2-NH2 for the next coupling reaction, respectively.

A solution of HCTU (2 mmol) and DIEA (4 mmol) in DCM/DMF (1:1) was added to a mixture of resin-*D*-Phe4-*L*/*D*-Val3-*D*/*L*-Val2-NH2, Fmoc-*L*-Trp (Boc)-OH (2 mmol). After the reaction mixture was stirred for 60 min, the resin was filtered and washed. To remove the Fmoc group, the same procedure was repeated as above. The resin was used as resin-*D*-Phe4-*L*-Val3-*D-*Val2-*L*-Trp1(Boc)-NH2/*D*-Phe4-*D*-Val3-*L*-Val2-*L*-Trp1(Boc)-NH2, respectively. Subsequent to TFA cleavage (TFA: TIPS: H2O, 90:5:5, 3 h), the reaction solution was treated with cold ether to obtain precipitation. The precipitate was furtherly purified by HPLC to give the compounds **1a** (*L*-Trp1-*L*-Val2-*D*-Val3-*D*-Phe4) and **1b** (*L*-Trp1-*D*-Val2- *L*-Val3-*D*-Phe4), respectively.

### *3.8. Xenopus Laevis Oocyte Preparation and Microinjection*

All procedures were approved by the University of Wollongong Animal Ethics Committees (project number: AE2003). Female *Xenopus laevis* were sourced from Nasco (Fort Atkinson, WI, USA), and a maximum of four frogs were kept in a 15 L aquarium at 20–26 ◦C with 12 h light/dark cycle. Oocytes were obtained from five-year-old frogs anesthetized with 1.7 mg/mL ethyl 3-aminobenzoate methanesulfonate (pH 7.4 with NaHCO3). Stage V-VI oocytes (Dumont's classification; 1200–1300 μm diameter) were defolliculated with 1.5 mg/mL collagenase Type II (Worthington Biochemical Corp., Lakewood, NJ, USA) at room temperature for 1–2 h in OR-2 solution containing (in mM): 82.5 NaCl, 2 KCl, 1 MgCl2 and 5 HEPES at pH 7.4.

The human muscle nAChR clones (*α*1, *β*1, *γ*, *δ* and *ε*) were purchased from Integrated DNA Technologies (Coralville, IA, USA), whereas the human *α*3, *α*9, *α*10, *β*2, and *β*4 clones were purchased from OriGene (Rockville, MD, USA), and all were subsequently inserted into the pT7TS vector. The human *α*4 and *α*7 clones were obtained from Prof. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA, USA). Plasmid constructs of the human nAChR clones were linearized for in vitro mRNA synthesis using mMessage mMachine transcription kit (AMBION, Forster City, CA, USA).

Oocytes were injected with 5 ng cRNA for h*α*1*β*1*γδ*, h*α*1*β*1*εδ*, h*α*3*β*2, h*α*3*β*4 and h*α*4*β*2, 10 ng cRNA for h*α*7 nAChR, and 35 ng cRNA for h*α*9*α*10 nAChR (concentration confirmed spectrophotometrically and by gel electrophoresis). The muscle subunit cRNA ratio was 2:1:1:1 (*α*1:*β*1: *γ*/*ε*:*δ*), whereas the heteromeric *α* and *β* subunit cRNA ratio was 1:1, injected using glass pipettes pulled from glass capillaries (3-000-203 GX, Drummond Scientific Co., Broomall, PA, USA). Oocytes were incubated at 18 ◦C in sterile ND96 solution composed of (in mM): 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES at pH 7.4, supplemented with 5% fetal bovine serum, 50 mg/L gentamicin (GIBCO, Grand Island, NY, USA) and 10,000 U/mL penicillin-streptomycin (GIBCO).
