Ctn (KRFKKFFKKVKKSVKKRLKKIFKKPMVIGVTIPF), Ctn[15-34] (KKRLKKIFKKPMVIGVTIPF), and their six topoisomers (Table 1 ) were synthesized in C-terminal carboxamide form in a Liberty Blue instrument (CEM Corporation, Matthews, NC, USA) using Fmoc solid phase peptide synthesis (SPPS) at 0.1 or 0.25 mmol scale on Rink Amide ProTide resin. Side chain functionalities were protected with tert-butyl (Ser, Thr), tert-butyloxycarbonyl (Lys), and 2,2,4,6,7-pentamethyldihydrobenzo- furan-5-sulfonyl (Arg) groups. Four equivalents of Fmoc-L- or Fmoc-D-amino acid derivative and equivalent amounts of Oxyma Pure (Merck, Darmstadt, Germany) and N,N′-diisopropylcarbodiimide were used for the couplings, with N,N-dimethylformamide (DMF) as solvent. After chain assembly, full deprotection and cleavage from the resin were conducted with trifluoroacetic acid (TFA)/H2O/triisopropylsilane (95:2.5:2.5 v/v, 120 min, rt.). Peptides were precipitated with cold diethyl ether, dissolved in water, and lyophilized.
Liberty blue
Manufactured by CEM Corporation
Sourced in United States
The Liberty Blue is a high-performance laboratory equipment designed for precise chromatographic analysis. It features advanced technology to ensure accurate and reliable results. The core function of this product is to facilitate the separation, identification, and quantification of chemical compounds.
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Lab products found in correlation
Oxyma pure, by Merck Group (1 mentions)
Jupiter proteo, by Phenomenex (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
E2695, by Waters Corporation (1 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Waters 3100 mass detector, by Waters Corporation (1 mentions)
Jupiter c18 column, by Phenomenex (1 mentions)
Acquity hplc, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Microflex, by Bruker (1 mentions)
Clear amide resin, by Peptide Institute (1 mentions)
11 protocols using liberty blue
1
Synthesis of Ctn Peptides and Topoisomers
2
PNA-Peptide Conjugates Synthesis
3
Synthesis and Purification of Pif80 Peptides
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All chemicals were obtained from Acros unless stated otherwise. The target peptides with C-terminal amidation were synthesized by standard Fmoc method46 (link). The scale of synthesis was 0.1 mmol. Pif80-7 and Pif80-11 were synthesized manually, whereas Pif80-22 was synthesized by microwave-assisted synthesizer (Liberty Blue, CEM Corporation) with Fmoc-PAL-PEG-PS resin (Life Technology), 10% (vol/vol) piperazine with 0.1 M HOBt in 1:9 ethanol/NMP solution as deprotection agent, N,N′-Diisopropylcarbodiimide in DMF as activator, and ethyl 2-cyano-2-(hydroxyimino) acetate (Merck) in DMF as activator base. Single coupling was performed for all amino acids at 90 °C for 4 minutes. The deprotection steps were conducted for 5 minutes, two times at room temperature without microwave irradiation. After drying the resin with dichloromethane, the crude peptides were cleaved from the resin with a reagent mixture of 92.5% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% 3,6-dioxa-1,8-octanedithiol (Sigma Aldrich), and 2.5% DI water for 2.5 hours. The crude peptides were then precipitated in methyl tert-butyl ester precooled at −20 °C. Crude product was dried and then purified by high-performance liquid chromatography at room temperature with a Vydac C18 column, utilizing a water/acetonitrile gradient with 0.1% TFA.
4
Fmoc-based Solid-Phase Peptide Synthesis
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All peptide syntheses were performed using standard Fmoc-based solid-phase peptide synthesis (SPPS) protocols on an automated Microwave Peptide Synthesizer (Liberty Blue, CEM Corporation). TentaGel R RAM was used as a solid resin, and DIPEA, DIC and Oxyma pure were used as coupling reagents. 20% piperidine in DMF was used to cleave Fmoc, and N-acetylimidazole was used as a capping reagent. Deprotection was performed in a mixture of Trifluoroacetic acid/Phenol/Triisopropylsilane/H2O (88:5:5:2 v/v/v/v). Peptides were purified by HPLC using a semi-preparative Jupiter Proteo (Phenomenex) column C12 4 µm, 90 Å with solvents containing TFA as an additive. The trifluoroacetate counterion of all peptides was exchanged for the acetate on SPE columns. The peptides were characterized by HPLC and LCMS (Supplementary Data—Table 1S , 2S and Fig. 1S ).
5
Solid-Phase Synthesis of Peptide 7-mers
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The peptides were synthesized in-house by the standard solid-phase synthesis (SPS) protocol using the microwave-assisted peptide synthesizer Liberty Blue (CEM Corporation, Matthews, North Carolina, USA). The resins, amino acids, and Oxyma pure were obtained from the same vendor. Dimethylformamide (DMF), diisopropyl carbodiimide (DIC), 4-methyl piperidine, acetonitrile, and all other chemicals were purchased (Sigma-Aldrich, St. Louis, MO, USA). The synthesized 7-mers were cleaved from the resin using a cocktail containing 92.5% trifluoroacetic acid (TFA), 2.5% deionized water, 2.5% triisopropylsilane (TIS), and 2.5% dioxa-1,8-octane-dithiol (DODT). The cleaved peptides were precipitated by the addition of ether and then redissolved in a 1% acetonitrile-water mixture. The solutions were lyophilized to obtain a solid powder of the peptides with a percentage yield ranging between 34–66%. The purity for the peptides was checked by high-performance liquid chromatography (HPLC) on a Shimadzu instrument using a Luna 5 μm C18, 100 Å, 250 × 4.6 mm reverse-phase analytical LC column with a gradient of 5% to 70% acetonitrile in water (both containing 0.1% (v/v) trifluoroacetic acid). All the NMR sample tubes were vacuum-sealed to avoid the absorption of water molecules by the solvent with a ~26 mM concentration in deuterated dimethyl sulfoxide.
6
Microwave-Assisted Peptide Synthesis and Purification
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Peptides were produced as C‐terminal amides using a Liberty Blue automated microwave peptide synthesiser (CEM Corporation, Matthews, NC, USA), using standard fluorenylmethyloxycarbonyl‐mediated solid‐phase chemistry and NovaPEG rink amide resin (Merck, Kenilworth, NJ, USA). Following synthesis, peptides were cleaved from the resin by incubation with trifluoroacetic acid/water/triisopropylsilane/dimethoxybenzene (92.5 : 2.5 : 2.5 : 2.5) for 3 h followed by precipitation with ice‐cold diethyl ether. For kinetic experiments, lyophilised peptides were purified by reverse‐phase high‐performance liquid chromatography using a Vydac C18 column (Solvent A: 0.1% trifluoroacetic acid in H2O, Solvent B: 0.1% trifluoroacetic acid in acetonitrile). Sequences are given in Table S1 .
7
Synthetic Peptide Production and Purification
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Synthetic peptides were produced by standard 9-fluorenylmethyloxycarbonyl/tert-butyl strategy using peptide synthesizers P11 (Activotec) or Liberty Blue (CEM Corporation). Purity was assessed by reversed phase HPLC (e2695; Waters) and identity affirmed by nano-UHPLC (UltiMate 3000 RSLCnano) coupled online to a hybrid mass spectrometer (LTQ Orbitrap XL; both Thermo Fisher Scientific). Lyophilized peptides were purified by standard HPLC. For certain peptides, a pH titration with 0.1 M NaOH was performed using standard procedures. For in vitro assays, peptides were dissolved at 10 mg/ml in DMSO and diluted 1:10 in bidistilled H2O. Frozen aliquots were further diluted in cell culture medium and sterile filtered if necessary.
8
Microwave-Assisted Synthesis of Gallocin Peptides
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Peptides were synthesised by microwave-assisted solid phase peptide synthesis (MW-SPPS) performed on a Liberty Blue microwave peptide synthesizer (CEM Corporation. Mathews, NC, USA). Gallocin D1 was synthesized on a H-Cys(Trt)-HMPB)-ChemMatrix resin and Gallocin D2 was synthesised on H-Phe-HMPB-ChemMatrix resin (PCAS BioMAtrix Inc., Quebec, Canada). Crude peptide was purified using RP-HPLC on a Semi Preparative Jupiter Proteo C12 (10 × 250 mm, 4 µ, 90 Å) column (Phenomenex, Cheshire, UK) running acetonitrile 0.1% TFA gradients specific to the peptide of interest. Fractions containing the desired molecular mass were identified using MALDI-TOF-mass spectrometry in positive in linear mode and were pooled and lyophilized on a Genevac HT 4X lyophiliser (Genevac Ltd., Ipswich, UK).
9
Synthesis and Purification of Immunogenic Peptides
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Highly immunogenic peptides, i.e., NPB (22–50), NPW (220–250) and NPBWR1 (33–62) were synthesized in solid phase using the automated microwave-assisted peptide synthesizer Liberty Blue (CEM Corporation, Matthews, NC, USA) following the standard protocols for Fmoc/tBu strategy. The peptides were purified by semipreparative RP-HPLC on a Waters instrument (Separation Module 2695, detector diode array 2996) using a Phenomenex (Torrance, CA, USA) Jupiter column C18 (10 μm, 250 × 10 mm) at 4 mL/min with solvent systems A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN). The purity of the peptides was analyzed by analytical HPLC using a Waters ACQUITY HPLC coupled to a single-quadrupole ESI-MS (Waters 3100 Mass Detector) supplied with a BEH C18 (1.7 μm 2.1 × 50 mm) column at 35 °C at 0.6 mL/min with solvent systems A (0.1% TFA in H2O) and B (0.1% TFA in CH3CN).
Data were acquired and processed using MassLynx software (Waters, Milford, MA, USA). The analytical data are reported in detail inTable 4 .
Data were acquired and processed using MassLynx software (Waters, Milford, MA, USA). The analytical data are reported in detail in
10
Synthesis and Characterization of Bet v 1-Derived Peptides
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Six non‐IgE‐reactive and non‐allergenic Bet v 1‐derived peptides described by Focke et al.
12 (link) (P1: aa 1–24; P2: aa 30–59; P3: aa 50–79; P6: aa 75–104; P4: aa 110–139; P5: aa 130–160; TableS3 ) were produced by chemical synthesis using 9‐fluorenylmethoxycarbonyl (Fmoc) amino acid protection and HBTU coupling on a peptide synthesizer (Liberty Blue, CEM Corporation). Peptides were purified to >90% purity by high‐pressure liquid chromatography (HPLC) (Dionex UltiMate 3000; Thermo Fisher Scientific), and their molecular weights were checked by MALDI‐TOF mass spectrometry (Microflex, Bruker). For comparing peptide‐specific IgG reactivity to Bet v 1 and Mal d 1 by micro‐array analysis, seven Bet v 1‐derived peptides as described in
31 (link) and the corresponding Mal d 1 peptides were prepared and characterized as described above.
12 (link) (P1: aa 1–24; P2: aa 30–59; P3: aa 50–79; P6: aa 75–104; P4: aa 110–139; P5: aa 130–160; Table
31 (link) and the corresponding Mal d 1 peptides were prepared and characterized as described above.
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