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9 protocols using fmoc protected amino acids

1

Synthesis of Fmoc-Protected Peptide F6C11

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All Fmoc-protected
amino acids,
Rink Amide resin, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate were purchased from Anaspec, Inc. The peptide
F6C11 was prepared according to our previous paper.45 (link) All the other reagents were used as received.
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2

Organic Synthesis Reagents Protocol

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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. PIMT was purchased from Fitzgerald Industries (Cat. # 80R-1342), SAM was purchased from Sigma (a7007), and tris base from Fisher Bioreagents (BP152).
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3

Peptide Synthesis and Purification

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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. Trypsin was purchased from Sigma Aldrich (T0303). α-chymoTrypsin was also purchased from Sigma Aldrich (C-4129).
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4

Peptoid Synthesis via Microwave-Assisted SPPS

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All the Fmoc-protected amino acids were purchased from AnaSpec
(Fremont, CA). TentaGel resin was purchased from Rapp Polymere GmbH
(German). All other reagents were purchased from Sigma-Aldrich or
Alfa Aesar, unless otherwise specified. All of the chemical reagents
and solvents from commercial sources were used without further purification.
Five mL and 10 mL disposable reaction columns (Intavis AG) were used
as reaction vessels for solid-phase synthesis. Syntheses of peptiods
under microwave conditions were performed in a 1500 W microwave oven
(GE model JE 1860BH04) with 10% power. HPLC was carried out on Waters
systems equipped with Waters 1525 binary HPLC pumps and a 2487 dual
λ
absorbance detector, or a 2998 photodiode array detector. The mobile
phase comprised of buffer A (H2O containing 20% CH3CN and 0.1% trifluoroacetic acid (TFA)) and buffer B (CH3CN containing 0.1% TFA). Analytical HPLC was conducted using
a Vydac C-18 column (5 μm, 250 × 4.6 mm, Alltech, Deerfield,
IL) at a flow rate of 1.0 mL/min with UV detection at 220 nm. MS and
MS/MS (MALDI-TOF) were performed on a 4800 Proteomics Analyzer (Applied
Biosystems) with α-cyano-4-hydroxycinnamic acid (CHCA) as a
matrix. All steps involving water utilized distilled water filtered
through a Barnstead Nanopure filtration system (Thermo Scientific)
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5

Organic Solvent-based Amino Acid Synthesis

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Organic solvents and reagents were purchased
from Fisher Scientific and Sigma-Aldrich and were used without further
purification. Fmoc-protected amino acids and Wang resins were purchased
from Anaspec Inc. or Chem-Impex International.
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6

Synthesis and Characterization of Peptide-PEG Hydrogels

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A fibronectin derived peptide with the sequence Cys-Arg-Gly-Asp-Ser (CRGDS), a scrambled version CRDGS and a lysine containing version CRGDSK for fluorescent labeling were each synthesized using standard solid phase Fmoc-peptide synthesis, which was performed using a 36S automated peptide synthesizer (CS Bio, Menlo Park, CA) on rink amide resin using Fmoc protected amino acids (AnaSpec, Fremont, CA). Peptides purity >90% was verified using MALDI-TOF MS and HPLC, while concentrations in solution were determined using Elman's reagent (Pierce) to quantify thiol content by comparing against a standard curve generated with L-cysteine (Sigma-Aldrich). PEG-norbornene (8-arm PEG-NB 20k) was synthesized using a hydroxyl-functionalized multi-arm PEG core (JenKem Technology, USA) as previously described in 5 detail 13 , 46 . Directly after the synthesis the PEG norbornene macromers were purified by dialysis in 3500 kDa cut off membranes in DI water. Purity ≥ 88% was confirmed using HNMR.
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7

Peptide Synthesis Using Commercial Reagents

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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.
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8

Radioisotope Generator and Radiolabeling Protocol

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224Ra/212Pb generator (VMT-a-GEN) was manufactured by Viewpoint Molecular Targeting, Inc. (Coralville, IA, USA) using 228Th stock obtained from the US Department of Energy Oak Ridge National Laboratory (Oak Ridge, TN, USA). 203PbCl2 was provided by the Cyclotron Facility at the University of Alberta (Edmonton, AB, Canada). 68GaCl3 was kindly provided by Dr. David Dick from the Department of Radiology at the University of Iowa (Iowa City, IA, USA). H-threoninol(But)-2-Cl-Trt-resin, Fmoc-protected amino acids, Fmoc-NH-PEG2-propionic acid and [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) were purchased from AnaSpec (Fremont, CA, USA). Instant thin layer chromatography (iTLC) paper was purchased from Agilent (Santa Clara, CA, USA). Pb and RE2 resins were provided by Eichrom Technologies (Lisle, IL, USA). Empty polypropylene SPE tubes with 20 μm PE frits were obtained from Millipore Sigma (Burlington, MA, USA). Sep-Pak® C18 cartridges were purchased from Waters Corporation (Milford, MA, USA). Other chemicals were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CD1-Elite SOPF mice were purchased from Charles River Laboratory (Wilmington, MA, USA). Athymic nude mice were obtained from Envigo (Indianapolis, IN, USA). SSTR2-positive rat pancreatic cancer cell line AR42J was purchased from ATCC (Manassas, VA, USA).
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9

Organic Solvent Purification and Reagent Use

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