Peptides were synthesized using a Protein Technologies SymphonyX synthesizer and using 4-methylbenzydrylamine hydrochloride resin (Iris Biotech GMBH) (Perez et al., 2018 ). Standard Fmoc-protected amino acid (AA) coupling occurred in the presence of 95 mM HCTU (Iris Biotech GMBH) and 200 mM N-methylmorpholine (Gyros Protein Technologies; S-1L-NMM) over two 20-minute coupling cycles. Fmoc deprotection occurred in the presence 20% piperidine in dimethylformamide (DMF, Iris Biotech GMBH;) over two 5-minute cycles. The peptides were purified to >95% purity by preparative C18 reverse phase HPLC (Agilent 1200 series) over a water/0.1% TFA and acetonitrile/0.1% TFA gradient characterized using HPLC-MS (Agilent 6300 MSD). Peptide substrates were dissolved in mass spectrometry grade water. Absorbance measurements at 280 nm wavelength were used to determine the peptide concentration; peptide extinction coefficients were calculated using ExPASy Bioinformatics Resource portal (https://web.expasy.org/protparam/ ).
N n dimethylformamide (dmf)
Manufactured by Iris Biotech
Sourced in Germany
The DMF (Dimethylformamide) is a versatile laboratory solvent that can be used in a variety of applications. It serves as a core component in various chemical reactions and processes within a laboratory setting. The DMF product offers a consistent quality and reliability that supports research and development activities.
Automatically generated - may contain errors
Lab products found in correlation
N methylmorpholine, by Gyros Protein Technologies (1 mentions)
Agilent 1200 series, by Agilent Technologies (1 mentions)
N n dimethylformamide (dmf), by Carl Roth (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Piperidine, by Biosolve (1 mentions)
Salicylaldehyde, by Merck Group (1 mentions)
Fmoc rink amide mbha resin, by Iris Biotech (1 mentions)
O 7 azabenzotriazol 1 yl n n n n tetramethyluronium hexafluorophosphate hatu, by Merck Group (1 mentions)
N n diisopropylethylamine dipea, by Iris Biotech (1 mentions)
Disodium hydrogen phosphate, by Merck Group (1 mentions)
Ethyl acetate, by Avantor (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Methanol, by Avantor (1 mentions)
Acetic acid, by Avantor (1 mentions)
Piperidine, by Iris Biotech (1 mentions)
Trifluoroacetic acid tfa, by Iris Biotech (1 mentions)
4 protocols using n n dimethylformamide (dmf)
1
Peptide Synthesis and Purification Protocol
2
Automated Peptide Synthesis and Purification
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Chemical synthesis of the peptides was carried out at the Faculty of Chemistry at the University of Wrocław (Poland). The synthesis of specific peptides was carried out manually in a syringe reactor (BRAUN Inject, Germany). Preloaded Wang resins (0.58–0.79 mM/g) (Iris Biotech GmbH) were used for the synthesis of fully protected peptides. Fmoc-protecting group (9-fluorenylmethyloxycarbonyl) was removed with 25 % piperidine solution (Sigma-Aldrich) in DMF (3 and 17 min) (Carl Roth GmbH + Co. KG). Amino acid coupling reaction was carried out using DMF as a solvent with the use of 3 equivalents TCTU (Iris Biotech GmbH) as the coupling reagent, three equivalents of HOBt [GL Biochem (Shanghai) Ltd.], and six equivalents of DIEA (Iris Biotech GmbH) as additives. The reactions were carried out for 150 min. Peptides were cleaved from the resin simultaneously with side chain deprotection using a mixture of TFA (Iris Biotech GmbH)/TIS (Alfa Aesar)/H2O (95: 2.5: 2.5, v/v/v). The reaction was carried out for 120 min. Then, the solution was transferred into cold diethyl ether (Sigma-Aldrich). The crude residue was collected, dissolved in water, lyophilized and purified by reversed-phase high-performance liquid chromatography. All peptides were obtained as their trifluoroacetate salts.
3
Fmoc-based Solid Phase Peptide Synthesis Protocol
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All protected AAs, Fmoc‐Rink amide MBHA resin, N‐(9‐fluorenylmethyloxycarbonyloxy)‐succinimide (Fmoc‐OSu), N,N‐dimethylformamide (DMF), 1‐methyl‐2‐pyrrolidinone (NMP), dichloromethane (DCM), diethylether (Et2O), 2,2,2‐trifluoroethanol (TFE), and N,N‐diisopropylethylamine (DIPEA) were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Pseudoproline dipeptides, O‐(7‐azabenzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium‐hexafluorophosphate (HATU), H‐Gly‐2‐Cl‐trityl resin, H‐Thr(tBu)‐2‐Cl‐trityl resin, Fmoc‐Gly‐NovaSyn‐TGT resin, Fmoc‐Dbz‐NovaSyn‐TGR resin, 4‐nitrophenylchloroformate, and disodiumhydrogenphosphate were from Merck (Darmstadt, Germany). 1‐Hydroxybenzotriazole (HOBt), 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU), N,N′‐diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), and piperidine were obtained from Biosolve (Valkenswaard, The Netherlands). HPLC‐grade acetonitrile (ACN), thiophenol, 4‐mercaptophenylacetic acid (MPAA), triisopropylsilane (TIS), thioanisole (TIA), tris(2‐carboxyethyl) phosphine hydrochloride (TCEP), guanidine hydrochloride (GuHCl), and salicylaldehyde were obtained from Sigma Aldrich (Vienna, Austria). HPLC‐grade trifluoroacetic acid (TFA) was from Alfa‐Aesar (Karlsruhe, Germany).
4
Solid-Phase Peptide Synthesis Reagents
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Fmoc-amino acids were purchased from Orpegen Peptide Chemicals (Heidelberg, Germany) and Novabiochem (Schwalbach, Germany). HBTU, resins, and further chemicals for solid-phase peptide synthesis, including reagent-grade N-methylmorpholine, piperidine, trifluoroacetic acid (TFA), and N,N-dimethylformamide (DMF) were purchased from IRIS Biotech (Marktredwitz, Germany), Sigma-Aldrich Chemie GmbH (Munich, Germany), Alfa Aesar (Karlsruhe, Germany), Abcr GmbH (Karlsruhe, Germany), VWR International (Darmstadt, Germany), and FLUKA Chemika (Seelze, Germany). Solvents (acetonitrile, water, methanol, ethyl acetate, diethyl ether) and chemicals (iodine, acetic acid) used for peptide purification and oxidation were obtained from VWR International (Darmstadt, Germany) and Fisher Scientific (Schwerte, Germany).
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