Fmoc amino acids

Tissue culture media and reagents were purchased from Life Technologies. Native GLP-1(7-37)-OH, glucagon, BMS21, compound 2, ¹²⁵I-GLP-1 and ¹²⁵I-glucagon tracer were synthesized by Novo Nordisk A/S. GIP and GLP-2 were purchased from Bachem. All other laboratory reagents were obtained from Sigma-Aldrich unless otherwise specified. BMS21 was assembled on rink-amide (0.57 mmol/g) at a 0.25 mmol scale using manual Fmoc chemistry. Fmoc amino acids were purchased from Protein Technologies or Novabiochem, Fmoc-(S)-2′-methyl Biphenylalanine, Fmoc-(S)-2′-ethyl-4′-methoxy biphenylalanine and Fmoc-(S)-2-fluoro-R-methylphenylalanine were synthesized as described previuosly18 (link). Loading of Fmoc-(S)-2′-methyl Biphenylalanine to the Rink amide resin was conducted using 5 eq aminoacid preactivated with N,N’-Diisopropylcarbodiimide, 1-Hydroxy-7-azabenzotriazole for 30 minutes and coupled to the resin overnight. The following aminoacids were coupled using 4 eq Fmoc amino acids preactivated with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-Hydroxy-7-azabenzotriazole for 30 minutes followed by coupling for 2 hours. Cleavage and purification was done similar described previously18 (link).

msPapA was prepared by solid phase peptide synthesis using either a Prelude peptide synthesizer (Protein Technologies Inc) or a Chorus peptide synthesizer (Protein Technologies Inc). The syntheses and subsequent purification followed the same procedure as reported (40 (link)). All of the Fmoc-amino acids were purchased from Protein Technologies Inc. The msPapA was quantified by dry weight and the Y17W msPapA was quantified by its absorbance at 280 nm.

All Fmoc-amino acids and Oxyma Pure were purchased from Protein Technologies (now Gyros Technologies) or Novabiochem, Merck. Fmoc-L-Glu-Otbu (γGlu), Fmoc-L-Lys(Mtt)-OH and Fmoc-L-Lys(ivDde)-OH were obtained from Iris biotech (Germany). Dimethylformamide (DMF) was from Solvias (Switzerland). Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH) was purchased from Flamma Group, Italy. 20-(tert-Butoxy)-20-oxooctadecanoic acid (C20-diacid), 18-(tert-Butoxy)-18-oxooctadecanoic acid (C18-diacid), (16-(tert-Butoxy)-16-oxooctadecanoic acid (C16-diacid) and 14-(tert-Butoxy)-14-oxooctadecanoic acid (C14-diacid) were purchased from Solvias. Fmoc-PAL-resin, trifluoroacetic acid, diethylether, piperidine, acetonitril, hydrazine and triisopropylsilan were from Merck. 5(6)-TAMRA (AS-81120) was purchased from Anaspec (USA). All buffers and salts for in vitro were purchased from Sigma Aldrich. ActOne assay: Human embryonic kidney (HEK) 293 cells stably expressing the cAMP sensitive calcium channel and one of the human Y1R, Y2R, and Y4R (Cat # CB 80300-244, CB 80300-245, CB 80300-270, Codex Bio-solution, Gaithersburg, MD, USA). SPA assay: CHO cells stably expressing the human Y₂R (Cat # CG1274, Multispan, Hayward, CA, USA).