Wang resin

Organic solvents and reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Acros Organics and used without further purification. FMOC-protected amino acids and Wang resins were purchased from Anaspec, Inc or Chem-Impex International. Glu isomers were purchased from CarboSynth.

All amino acids for peptide synthesis as well as HBTU and Wang resin were purchased from Anaspec. Solvents and reagents for peptide synthesis and isolation including dichloromethane (DCM), N,N-dimethylformamide (DMF), N-methylmorpholine, trifluoroacetic acid (TFA), methyl tert-butyl ether (MTBE) and toluene were obtained from Fisher Scientific. Piperidine was obtained from Sigma-Aldrich. Homobifunctionalized carboxylated telechelic linear- and star-PEG derivatives of varying molecular weights including linear PEG 2,000, linear PEG 5,000, 4-arm PEG 10,000, and 4-arm PEG 20,000 were all purchased from JenKem Technology USA. Deionized water (18 MΩ, Direct-Q, Millipore, Billerica, MA) along with reagent alcohol (90:5:5 vol % (ethanol:methanol:isopropanol) was used to clean all glassware prior to experiments. Pluronic F108 was donated from BASF Global. Red fluorescent 1.0 micron spheres were purchased from Invitrogen. Peptide synthesis was carried out on a Protein Technologies Inc. PS3 peptide synthesizer.

Peptides NYLETTSDF, NYLETTSDFHF and CYGETYQNI were synthesized by Fmoc solid phase methods on preloaded Wang resin (Anaspec, Inc.) by Mayo Clinic’s Medical Genome Facility’s Peptide Synthesis Services. Each peptide chain was assembled from Nα-Fmoc protected amino acids on a Liberty Blue Microwave-Assisted Peptide Synthesizer (CEM Corp.) according to the manufacturer’s coupling and deprotection protocols. After synthesis each peptide was deprotected and removed from its resin support by acidolysis with a solution of trifluoroacetic acid containing 2.5 % water (v/v) and 2.5% triisopropylsilane (v/v) and 2.5% 3,6, dioxa-1,8-octanedithiol (v/v) for 30min at 42C. The crude peptide was then purified by preparative RP-HPLC using an aqueous acetonitrile gradient containing 0.1 % TFA (v/v) on a reverse-phase C18 column (Phenomenex Jupiter 15µ; 250 × 21.2 mm). The mass weight was verified by LC-ESI mass spectrometry on an Agilent 6224 TOF LC/MS instrument. Peptide homogeneity was confirmed by analytical RP-HPLC and was 99.9%, 95.5%, and 98.2% for NYLETTSDF, NYLETTSDFHF and CYGETYQNI respectively.

Peptides were synthesized using the stepwise solid-phase method by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on Wang resin (AnaSpec, Fremont, CA, USA) with a 12-channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ, USA) according to the manufacturer’s procedures. Detachment of peptide from the resin and removal of the side chain protection groups were done by incubating the resin with a mixture of trifloroacetic acid (TFA):Thioanisole:Water:Phenol:1,2-ethanedithio (82.5:5:5:5:2.5 v/v) for 2 hours.
The crude peptide was purified on a preparative Kinetex reversed-phase C18 column, 150 × 21.1 mm (Phenomenex, Torrance, CA, USA) using a BioCad Sprint (Applied Biosystems, Foster City, CA, USA). A flow rate of 30 mL/min with solvent A (0.1% TFA in deionized water) and solvent B (0.1% TFA in acetonitrile) was used. The column is equilibrated with 5% solvent B before sample injection. Elution is performed with a linear gradient from 5% solvent B to 100% solvent B in 60 min. The absorbance of the column effluent is monitored at 214 nm, and peak fractions are pooled and lyophilized.
The pure peptide fraction is identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or electrospray ionization mass spectrometry (ESI-MS) and lyophilized.

Peptide was synthesized using a stepwise solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on a Wang resin (AnaSpec, Fremont, CA) with a 12 channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ) as described previously (26 ). Briefly, peptide synthesis started from the C-terminus. The Fmoc group of the resin was removed with 20% piperidine in N, N-Dimethylformamide (DMF) (5 min x2) followed by washing the resin with DMF (6.5 min). After completion, the N-terminal Fmoc was removed with 20% piperidine in DMF (5 min x 2) followed by washing the resin with DMF (6.5 min). Resins were mixed with 5 mL 50% chloroform in DMF containing 5 g myristic anhydride and kept at 60°C for 1 hr. The crude peptide was then purified on a preparative Kinetex reversed-phase C18 column (Phenomenex, Torrance, CA) using a BioCad Sprint analyzer (Applied Biosystems, Foster City, CA). Elution was performed with a linear gradient from 5% solvent B to 100% solvent B in 30 min. The absorbance of the column effluent was monitored at 230 nm and peak fractions were pooled and lyophilized. The pure peptide fraction was identified by electrospray ionization mass spectrometry (ESI-MS) and lyophilized. Finally, peptides were dissolved in DMSO and 10 mM aliquots stored at −80°C.