In brief: Peptides were synthesized on solid support using a fully automated microwave peptide synthesizer (Liberty, CEM Corporation). The peptides were synthesized by solid phase peptide synthesis (SPPS) methodology67 with a fluorenylmethoxycarbonyl (Fmoc)/tert-Butyl (tBu) protocol. The lysine side chain was protected with N-methyltrityl (Mtt), a protection group that can be deprotected selectively using acid labile conditions.68 (link) After completion of the synthesis of the linear peptide, an anhydride spacer was coupled to the N-terminal amino group and cyclization was performed using amide bonds between the moiety linker at the backbone N-terminus and an epsilon amino on the side chain of a C-terminal Lys residue,69 (link),70 (link) The final cleavage and side chain deprotection was done manually without microwave energy. Peptides were analyzed by analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) (Shimadzu, MD, USA) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and purified by preparative RP-HPLC (Shimadzu, MD, USA). For full details, see supporting information .
Preparative rp hplc
Manufactured by Shimadzu
Sourced in United States, Japan
The Preparative RP-HPLC is a high-performance liquid chromatography system designed for the purification and separation of compounds on a preparative scale. It utilizes reversed-phase chromatography principles to achieve efficient separation and recovery of target analytes from complex mixtures.
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2 protocols using preparative rp hplc
1
Cyclic Peptide Synthesis by SPPS
2
Ginsentide Purification from Medicinal Plants
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Ginsentide TP1, TP3 or TP8 were extracted from the dried flowers or seeds of P. ginseng, P. quinquefolius, and P. notoginseng as previously described18 . Briefly, the plant materials were pulverized and extracted with water. The extracts were filtered and subjected to flash chromatography using C18 powder (Grace Davison). 60% ethanol was used to elute the ginsentide-enriched fractions which were then loaded onto an SP Sepharose resin column (GE Healthcare, UK) and eluted with 1 mol/L NaCl (pH 3.0). Further purification was done using preparative RP-HPLC (Shimadzu, Japan) with a C18 Grace Vydac column (250 mm × 22 mm) at a flow rate of 8 mL/min, linear gradient of 1%/min of 10%–80% buffer B. Buffer A contained 0.05% (v/v) trifluoroacetic acid (TFA) in HPLC grade water, and buffer B contained 0.05% (v/v) TFA and 99.5% (v/v) acetonitrile (ACN). Resulting fractions were further purified using a semi-preparative C18 Vydac column (250 mm × 10 mm) at a flow rate of 3 mL/min with the same linear gradient. The identity of the ginsentide containing fractions were confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS; AB SCIEX 5800 MALDI-TOF/TOF).
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