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Large-scale synthesis of peptides.
Since the specificity for the acyl donor and the nucleophile linkage are individual parameters for each enzyme, the efficiency of synthesis and the potential for peptide synthesis will greatly differ from one protease to another.
For SPPS of the Lys(Tct)-Lys-Lys-Pro-Lys-Lys-Arg-Lys-Val-Cys-OH [Lys(Tct)-NLS(SV40-T)-Cys] and the Cys-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-OH (Cys-pAnt43-58), the Fmoc-strategy was applied in a fully automated multiple synthesizer. The synthesis was carried out in a 0.05 mmol scale on a Fmoc-Lys(Boc)-polystyrene resin (1% cross-linked) with 0.053 mmol/g loading and Fmoc-Cys(Trt)-polystyrene resin (1% cross-linked) with 0.005 mmol/g loading. As coupling reagents, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HBTU)/HOBt/DIPEA (1:1:1) was used. Cleavage/deprotection of the peptide-resin was performed with TFA/ethane dithiol/thioanisole/phenol for 2.5 h at room temperature. The products were precipitated with ether. The crude products were purified by preparative HPLC on a Kromasil 300-5C18 reverse phase column (20 × 150 mm) using as eluents 0.1% TFA in water (A) and 60% acetonitrile in water.
Proteases in organic synthesis.
The next (Nα protected) amino acid is coupled to the already synthesized peptide chain bound to the polymeric matrix and, once coupled, its Nα amino group is deprotected.
Solid supports should meet several requirements: particles should be of conventional and uniform size, mechanically robust, easily filterable, chemically inert and chemically stable under the conditions of synthesis and highly accessible to the solvents allowing the penetration of the reagents and the enlargement of the peptide chain within its microstructure.
Synthesis of proteins by native chemical ligation.
Despite these drawbacks, solid-phase synthesis has many advantages over the classical system in solution: the reaction can be automated and the problem of solubilization of the peptide no longer exists since it remains attached to the solid matrix.
The PNA-sequences for hybridization to the different ORI-target sequences of the phNIS-IRES-EGFP were identified. The syntheses of the peptide modules and the PNA were carried out by Solid Phase Peptide Synthesis in a fully automated synthesizer. For PNA synthesis, we used fluorenylmethoxycarbonyl (Fmoc)-protected monomers with the exocyclic amino groups of A, G, and C bases blocked by a benzhydroxyl (Bhoc) group. Sequences of single modules as well as the complete modular construct were characterized with analytical HPLC and laser desorption mass spectrometry. Myristic acid was coupled with tetramethylfluoroformamidium-hexafluorophosphate (TFFH) in dimethylformamide/dichloromethane for one hour at the N terminus of PNA. Cysteine groups were attached via one of the COOH-terminal lysine residue of pAntp(43-58)-Cys and at the NH2-terminus of (NLS[SV40-T]). Molecules were oxidized in an aqueous solution of 2mg/ml in 20% DMSO for about five hours. The oxidation progress was monitored by analytical C18 reverse-phase HPLC. Peptide nucleic acids as well as the address peptide (NLS) carried one lysine-lysine spacer at the COOH terminus, which enabled linkage of peptide nucleic acids with identical sequence via a succinimidyl ester in a molar ratio of 1:1.
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Synthesis of native proteins by chemical ligation.
When Merrifield introduced the method of solid-phase synthesis in 1963, the scientific community reacted with skepticism: the synthesis in solution was at that time well established and in the new proposed system the purification of the peptide could only be done after cleavage, with the concomitant cleavage of most of the byproducts accumulated during the synthesis (
Modified proteinases in peptide synthesis in organic media.
Once the peptide synthesis of the desired sequence is finished, the protecting groups of the side chains are removed and the peptide freed from the support.
A side reaction in solid-phase peptide synthesis.
Its main advantage is that the intermediate products can be isolated and purified after each step of synthesis, deprotected and recombined to obtain larger peptides of the desired sequence.
Solid-phase synthesis of b-oligopeptides.
However, larger size polypeptides can be constructed using sequential synthesis by the technique of cysteine polymerization, the construction of dendrimers using lysine matrices, or the construction of Template-Assembled Synthetic Protein (TASP) (;
Synthesis of peptide bonds by proteinases.
The synthesis of the peptide with DDZ (dimethyl-3,5-dimethoxybenzyloxycarbonyl) protected-amino acid was carried out in solution. The peptides were synthesized twice on a 0.5% cross-linked polystyrene gel. All the synthetic steps were functionally controlled photometrically. Instead of Gln and Asn, Glu(OBz1) and Asp(OBz1) were used in order to avoid nitrile formation at the amide side by the condensation reagent dicyclohexylcarbodiimide (DCC). Asp was linked to the carrier with its β-carboxyl function via an electrophilic 2-oxoethyl ester bond. In this way all the later amide sides could be incorporated simultaneously at the end of the synthesis by ammonolysis of the benzyl ester and 2-oxoethyl ester bonds on release from the carrier. Proof of the stability of the benzyl ester in position A under the cleavage conditions was provided by the mass spectrum.
Proteases in peptide synthesis.
In convergent synthesis, peptides (up to 50 residues) are separately produced by sequential synthesis and then linked in solution or in solid phase to obtain the desired high molecular weight peptide or protein.
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