Fig. 6: A : ESI+ MS of benzylamine pentameric peptoid

1. Peptoid Preparation on the CEM Manual Peptide Synthesizer Adrian S. Culf 1,2*, Jennifer A. Melanson 1,2, Daniel A. Léger 1, Rodney J. Ouellette 1 1. Atlantic Cancer Research Institute, 35 Providence St., Moncton, NB. E1C 8X3 Canada 2. Department of Chemistry and Biochemistry, Mount Allison University, Sackville, NB. E4L 1G8 Canada *Corresponding author: adrianc@canceratl.ca; aculf@mta.ca Fibre-optic probe open microwave cavity Introduction α-Peptoids, or N-substituted glycine oligomers, have been synthesized by the sub-monomer method (Scheme 1) in a variety of experimental apparatuses, since 1992 [1,2]. Although most syntheses are conducted at room temperature, considerable time saving can accrue upon controlled heating. Purity increases can also be observed. Scheme 1 Domestic [3] as well as commercial multimode microwave synthesizers [4,5] are used for peptoid synthesis, although precise temperature control, where used, involves the construction of custom equipment [4]. The use of monomode instrumentation has allowed the measurement of temperature using a remote IR probe [6,7]. We have used the CEM Manual Peptide Synthesizer [Fig.1, 8], equipped with a fibre-optic temperature probe and vacuum suction station for the solid-phase synthesis of α-peptoids in a polypropylene tube. vacuum station solvent waste Fig 1: CEM Manual Peptide Synthesizer PTFE casing Fig 7: Other peptoids currently being used in cell culture assay qualification PP tube reaction vessel PP tube reaction vessel vacuum manifold Fig 3: Vacuum assisted draining and washing of resin beads Fig 2: Resin beads and reactants are contained in a polypropylene syringe body – ball valve in the nozzle. B A Conclusions The monomode microwave CEM Manual Peptide Synthesizer has been used for the first time to synthesize α-peptoid oligomers on PS-Rink amide solid support resin. Acknowledgements We thank the New Brunswick Student Employment Experience Development (NB-SEED) program and the funding sources of ACRI. We also thank Martin Léger (Environment Canada, Moncton, NB) for the use of a lyophilizer. Fig 4: Peptoid pentamer used as a model system for synthesis optimization • References • Zuckermann, R.N.; Kerr, J.M.; Kent, S.B.H.; Moos, W.H. “Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis.” J. Am. Chem. Soc.1992, 114, 10646-10647. • Culf, A.S.; Ouellette, R.J. “Solid-phase Synthesis of N-substituted Glycine Oligomers (α-peptoids) and Derivatives” Molecules 15, xxx, (2010). Accepted and in press. • Olivos, H.J.; Alluri, P.G.; Reddy, M.M.; Salony, D.; Kodadek, T. “Microwave-Assisted Solid-Phase Synthesis of Peptoids.” Org. Lett. 2002, 4, 4057-4059. (1kW Whirlpool domestic - multimode) • Gorske, B.C.; Jewell, S.A.; Guerard, E.J.; Blackwell, H.E. “Expedient Synthesis and Design Strategies for New Peptoid Construction.” Org. Lett.2005, 7, 1521-1524. (Milestone MicroSYNTH - multimode) • Seo, J.; Michaelian, N.; Owens, S.C.; Dashner, S.T.; Wong, A.J.; Barron, A.E.; Carrasco, M.R. “Chemoselective and microwave-assisted synthesis of glycopeptoids.” Org. Lett. 2009, 11, 5210-5213. (CEM Mars – multimode) • (a) Messeguer, J.; Cortes, N.; Garcia-Sanz, N.; Navarro-Vendrell, G.; Ferrer-Montiel, A.; Messeguer, A. “Synthesis of a positional scanning library of pentamers of N-alkylglycines assisted by microwave activation and validation via the identification of trypsin inhibitors.” J. Comb. Chem.2008, 10, 974-980. (b) Nnanabu, E.; Burgess, K. “Cyclic semipeptoids: Peptoid-organic hybrid macrocycles.” Org. Lett.2006, 8, 1259-1262. (CEM Discover - monomde) • Fara, M.A.; Diaz-Mochon, J.J.; Bradley, M. “Microwave-assisted coupling with DIC/HOBt for the synthesis of difficult peptoids and fluorescently labeled peptides-a gentle heat goes a long way.” Tetrahedron Lett. 2006, 47, 1011-1014. (Biotage SmithSynthesizer - monomode) • www.cem.com Fig 5: HPLC of benzylamine pentameric peptoid. Zorbax XDB-C8 column, 0.5 mL/min. Linear gradient of 1:1 acetonitrile(0.01% TFA): water(0.01% TFA) to 100% acetonitrile(0.01% TFA) over 15 mins. UV detection at 214 nm. Arrow indicates peptoid pentamer. Methods Fmoc Deprotection: 4-methylpiperidine (20% in DMF, 12.5 mL) was added to PS-Rink amide resin (Novabiochem, 100mg, 0.6 mmol/g, 60 μmoles) with stirring for 30 mins. at rt. The resin was washed with DMF, iPrOH, DCM (3x2mL each solvent). The procedure was repeated and the deprotected resin was visualized with bromophenol blue indicator (5 drops, 0.05% DMA solution). All the solvents used were anhydrous with DMF being additionally amine free (Caledon Laboratories). Acylation reactant: A stock solution of bromoacetic anhydride was prepared from bromoacetic acid (1 eq.) and DIC (1 eq.) in anhydrous DCM (0.12 mmol/mL) for each synthesis session. Insoluble white urea byproduct was filtered off before use. Acylation reaction: Deprotected PS-Rink amide resin (100mg, 0.6 mmol/g) and bromoacetic anhydride DCM solution (7 or 20 eq.) were reacted at 35 oC for 60 s (150 W) with stirring and direct fibre-optic temperature control of the reaction mixture. The resin was washed with DMF, iPrOH, DCM (3x2mL each solvent). Completion of reaction was checked with bromophenol blue indicator (5 drops, 0.05% DMA solution; yellow). Amination reaction: DMF (6 mL) and a primary amine (20 or 70 eq.) was reacted with the acylated resin at 90 oC for 120 s (150 W) with stirring and direct fibre-optic temperature control of the reaction mixture. The resin was washed with DMF, iPrOH, DCM (3x2mL each solvent). Completion of reaction was checked with bromophenol blue indicator (5 drops, 0.05% DMA solution; blue). Cleavage from resin: Peptoids were cleaved from the resin using TFA:water:TIS (95:2.5:2.5 (v/v), 1 mL) for 2 hours at rt. The suspension was filtered and washed through with a further aliquot of cleavage cocktail (1 mL) and water (2x 1 mL), then frozen at -80 oC for 90 mins. The peptoid was lyophilized (Labconco freeze dryer) for 16 hours, redissolved in acetonitrile:water (1:1 (v/v)) and re-lyophilized to yield a white powder. See table for yields and purities observed. Fig. 6: A: ESI+ MS of benzylamine pentameric peptoid showing expected m/z 753 molecular ion. B: MS/MS mass spectrum showing expected B and Y fragment ions.