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5 protocols using 2 1h benzotriazol 1 yl 1 1 3 3 tetramethyluronium hexafluorophosphate hbtu

1

Solid-Phase Peptide Synthesis Protocol

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Tentagel macrobeads were purchased from Rapp Polymere (Germany). All the Fmoc protected amino acids, 2‐(1H‐benzotriazol‐1‐yl)−1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU), and HOBt were purchased from EMD millipore (Billerica, MA). All primary amines, bromoacetic acid, diisopropylcarbodiimide (DIC), N,N‐diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), and dimethyl formamide (DMF) were purchased from Sigma‐Aldrich (Milwaukee, WI); Dichloromethane (DCM) and acetonitrile were obtained from Honeywell Burdick & Jackson (Morristown, NJ, USA). H‐β‐Ala‐OtBu.HCl was purchased from EMD Biosciences (Gibbstown, NJ, USA). All of the chemical reagents and solvents from commercial sources were used without further purification. 5 mL disposable reaction columns (Intavis AG) were used as reaction vessels for solid‐phase synthesis.
MALDI‐TOF mass spectra were acquired on an Applied Biosystems Voyager‐6115 and Voyager De‐Pro using α‐Cyano‐4‐hydroxycinnamic acid as matrix, while MS/MS (MALDI‐TOF) for sequencing were performed on a 4700 Proteomics Analyzer (Applied Biosystems).
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2

Solid-Phase Synthesis of Peptides

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Tentagel macrobeads were purchased from Rapp Polymere (Germany). All the Fmoc protected amino acids, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and HOBt were purchased from EMD millipore (Billerica, MA). All primary amines, bromoacetic acid, diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIPEA), piperidine,, trifluoroacetic acid (TFA), and dimethyl formamide (DMF) were purchased from Sigma-Aldrich (Milwaukee, WI, USA); Dichloromethane (DCM) and acetonitrile were obtained from Honeywell Burdick & Jackson (Morristown, NJ, USA). H-β-Ala-OtBu.HCl was purchased from EMD Biosciences (Gibbstown, NJ, USA). All of the chemical reagents and solvents from commercial sources were used without further purification. 5mL disposable reaction columns (Intavis AG) were used as reaction vessels for solid-phase synthesis.
MALDI-TOF mass spectra were acquired on an Applied Biosystems Voyager-6115 and Voyager De-Pro using α-Cyano-4-hydroxycinnamic acid as matrix, while MS/MS (MALDI- TOF) for sequencing were performed on a 4700 Proteomics Analyzer (Applied Biosystems).
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3

Solid-Phase Peptide Synthesis Methodology

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Acetic acid, acetone, acetonitrile, Fmoc protected Amino acids, Bis-Boc-aminooxy acetic acid (Aoa), Dichloromethane (DCM), N,N-Diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), Ethyl cyano(hydroxyimino)acetate (Oxyma Pure), Methanol, Rink-Amide MBHA resin, Triethylamine (TEA), Triethylsilane (TES), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU) were purchased from Merck Millipore (Burlington, MA, USA). Diethyl ether, Trifluoroacetin acid (TFA) were purchased from Biosolve (Valkenswaard, Holland). 2-Proponal for HPLC (aldehyde and ketone free) was purchased from PanReac AppliChem (Darmstadt, Germany). Ethanol was purchased from Carlo Erba (Milan, Italy). N-Methyl-2-pyrrolidone (NMP) was purchased from Iris Biotech GmbH (Marktredwitz, Germany).
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4

Peptide Synthesis and Chondrogenesis

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9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) protected amino acids, lauric acid, [4-[a-(2 0 4 0 -dimethoxyphe nyl) Fmoc-amino methyl] phenoxyl] acetomidonorleucyl-MBHA resin (Rink amide MBHA resin), Fmoc-Asp (OtBu)-Wang resin and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) were purchased from Merck, NovaBiochem, Sigma-Aldrich and ABCR. Cover glasses and tissue culture plates were purchased from NEST Biotechnology and Corning. Antibodies were obtained from Abcam. Chondrogenesis Differentiation Kit was purchased from Thermo Fisher Scientific. All other materials and chemicals used in the study were analytical grade and purchased from Invitrogen, Fisher, Merck and/or Sigma-Aldrich. All the chemicals were used as provided.
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5

Peptide Synthesis and Cell Culture Protocol

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9-Fluorenylmethoxycarbonyl (Fmoc) and other protected amino acids, lauric acid, [4-[a-(2 0 ,4 0 -dimethoxyphenyl)Fmocamino methyl]phenoxy] acetomidonorleucyl-MBHA resin (Rink amide MBHA resin), 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylethylamine (DIEA) were purchased from Merck. ABCR Collagenase V and Streptozotocin (STZ) were purchased from Sigma-Aldrich. RPMI 1640, Hanks Balanced Salt Solution (HBSS), Fetal Bovine Serum (FBS), penicillin/streptomycin/amphotericin B and L-glu- tamine were purchased from Lonza. Biocoll 1100 and Biocoll 1077 were purchased Biochrom. Rat-Mouse Insulin Assay and cell inserts were purchased from Milipore. VEGF was purchased from R&D and human FGF-2 from Sigma-Aldrich. All chemicals were used as provided.
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