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Peptide Synthesis and Characterization Stages
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
For the solid-phase synthesis of a synthetic peptide (NH2 - Ser - Leu - Thr - Trp - His - Lys - His - Glu - Leu - His - Arg - Lys - COOH), which has protective properties against Q fever disease, Wang resin loaded with serine amino acid, amino acids, and binding reagents is required. Furthermore, DMF, DCM, TFA, HOBt, HCTU, DIEA, NMP, and Fmoc-protected amino acids are also necessary substances for this solid-phase synthesis. 2- (6-chloro-1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole (HCTU/HOBt) was used as activator in DMF by microwave-assisted SPPS method for antigenic peptide synthesis to be synthesized (activator) and N,N-diisopropylethylamine/N-methyl – 2-pyrrolidinone (DIEA/NMP) activator base, piperidine was used as deprotector (DEP). Wang resin loaded with serine amino acid, was inflated by waiting in DMF for three hours before synthesis started. Another important step is the separation of the peptide from the resin (breaking, cleavage). This is the process of separating the peptide chain growing on the polymeric resin from this resin. The peptide cleavage cocktail [(TFA/thioanisole/EDT/water), (87.5/5/2.5/5 mL, (v/v)] obtained from the device depending on the resin and peptides shaken slow with a shaker at room temperature with the cleavage cocktail. Also, cold ether (−20°C) was used for precipitation and separation of the peptide. The centrifuged peptide precipitation procedure was repeated, washing the final precipitate, and dried. The dried peptide was stored at −18°C. Finally, the peptide synthesized in the purification and characterization phase of the peptide was determined by HPLC (Shimadzu). By using a Shimadzu PRC-ODS C-18 column 30 × 2.1 column it was analyzed with the aid of UV detector, and purification was carried out. A LC-MS system (Shimadzu LC-MS 2010 EV) Electro Spray Ionizer (ESI) probe and Teknokroma Tracer Exel 120 ODS-A, C-18 colon (20 cm length and 0.21 cm inlet diameter) and HPLC (4.6 × 250 mm, Venusil MP A gradient of 220 nm acetonitrile/water in a C18-5 column was used for characterization of the peptide. The isoelectric point of the peptide synthesized using Innovagen’s peptide was calculated: pH 10.66; Also, its net load at pH 7 was calculated as 2.3. In addition, it has been found to be well soluble in water and will be used in future vaccine prototype development studies; this is considered to be advantageous in conjugation with the polymer (Karahan 2017).
Nano-encapsulated HHC10 host defense peptide (HDP) reduces the growth of Escherichia coli via multimodal mechanisms
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Ankur Sharma, Kalpesh Vaghasiya, Eupa Ray, Rahul Kumar Verma
HHC10 peptide was synthesized on a Liberty microwave peptide synthesizer (CEM Corporation) using standard solid-phase FMOC protocols on Rink amide 4-methylbenzhydrylamine resins. The side-chains protecting group were removed and peptide was cleaved from the support-resin with a mixture of trifluoroacetic acid, thioanisole, ethane-di-thiol, and anisole (90% TFA 9 ml, 5% thioanisole 0.5 ml, 3% 1,2-ethanedithiol 0.3 ml, 2% anisole 0.2 ml) for 3 h. Synthesized peptide-resin mixture was filtered and repeatedly extracted with cold ether. Peptides were purified by semi-preparative RP-HPLC [(SunFire Preparative Column C18, 5 µm (10 × 250 mm)] on a WATERS HPLC system (Waters Corporation, Milford, MA) and confirmed by MALDI-TOF mass spectrometry (Applied Biosystems 4700 Proteomics Analyzer, Waltham, MA) [15].
Cyclic peptide-based nanostructures as efficient siRNA carriers
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Bijayananda Panigrahi, Rohit Kumar Singh, Sourav Mishra, Dindyal Mandal
The synthesis of amphipathic cyclic peptides was performed according to the previously reported procedure [35]. All reactions related to linear peptides were conducted in polypropylene columns by shaking and mixing using a vertical rotator at room temperature unless stated otherwise. Briefly, all linear peptides were made by SPPS strategy using Fmoc chemistry. HBTU and DIPEA in DMF were used as coupling and activating reagents, respectively. Fmoc deprotection at each step was done using piperidine in DMF (20% v/v). Side chain protected peptides were cleaved from the solid phase by shaking the resins with a mixture of trifluoroethanol (TFE)/acetic acid/dichloromethane (DCM) (2:2:6, v/v/v, 15 ml) for 2 h. The liquid was separated from resin by filtration and then evaporated by rotary evaporator to get dry side chain-protected linear peptides. The cyclization of the linear peptides was performed in the presence of HOAT and DIC in dry DMF (100 ml) and dry DCM (40 ml) for 24 h. Then, solvent was evaporated. The side chain deprotection was done with trifluoroacetic acid (TFA)/thioanisole/anisole/1,2-ethanedithiol (EDT) (90:5:2:3 v/v/v/v) for 2 h. The crude peptides were precipitated by the addition of cold diethyl ether (75 ml) and purified by Agilent 1260 infinity Quaternary LC system reversed-phase column (2.1 cm× 25 cm) using a gradient system.
Topoisomerases inhibition and DNA binding mode of daunomycin–oligoarginine conjugate
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Valeria Visone, Ildikó Szabó, Giuseppe Perugino, Ferenc Hudecz, Zoltán Bánóczi, Anna Valenti
Synthesis of Dau-Arg6 conjugate was performed as described by Miklan et al.14 with some modifications. Briefly, the synthesis of hexaarginine oligopeptide was carried out manually on Fmoc- Rink-Amide MBHA resin using standard Fmoc/tBu strategy. For coupling, Fmoc-Arg(Pbf)-OH and DIC/HOBt as coupling agents, were applied in DMF for in situ active ester formation. The N-terminal of the hexaarginyl-resin was functionalised by Boc-(aminoxy)acetic acid (Boc-Aoa-OH), using DIC/HOBt coupling reagents. The aminoxyacetylated peptide (Aoa-Arg6) was cleaved from the resin with TFA in the presence of scavengers (phenole, thioanisole). It should be noted that EDT was replaced by TIS and hydroxyl amine.