Solid Phase Peptide Synthesis a Practical Approach - …
Soybean lecithin consisting of 90–95%phosphatidylcholine and mPEG-DSPE, andMal-PEG-DSPE were purchased from Avanti Lipid(Alabaster, AL, USA). Cholesterol (CHO) was purchased from ChengduKelong Chemical Company (Chengdu, China). Rhodamine-PE waspurchased from Avanti Lipid. T7 peptide with terminal cysteine(Cys-HAIYPRH) and TAT peptide with terminal cysteine(Cys-AYGRKKRRQRRR) were produced according to the standard solidphase peptide synthesis by Shanghai Jier Bio-Pharmaceutical Co.,Ltd. (Shanghai, China). Cell culture plates were purchased fromWuxi NEST Biotechnology Co., Ltd. (Wuxi, China). Other chemicalsand reagents were of analytical grade and obtainedcommercially.
Solid Phase Peptide Synthesis : A Practical Approach by R
Functionalized proline residues have diverse applications. Herein we describe a practical approach, proline editing, for the synthesis of peptides with stereospecifically modified proline residues. Peptides are synthesized by standard solid-phase peptide synthesis to incorporate Fmoc-hydroxyproline (4-Hyp). In an automated manner, the Hyp hydroxyl is protected and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In a model tetrapeptide (Ac-TYPN-NH2), 4-Hyp was stereospecifically converted to 122 different 4-substituted prolyl amino acids, with 4 or 4 stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted prolines synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, nitrobenzoate), handles for heteronuclear NMR (19F:fluoro; pentafluorophenyl or perfluoro--butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence, IR: cyanophenyl ether), leaving groups (sulfonate, halide, NHS, bromoacetate), and other reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, 1,2-aminothiol). Proline editing provides access to these proline derivatives with no solution-phase synthesis. All peptides were analyzed by NMR to identify stereoelectronic and steric effects on conformation. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including azide–alkyne, tetrazine--cyclooctene, oxime, reductive amination, native chemical ligation, Suzuki, Sonogashira, cross-metathesis, and Diels–Alder reactions. These proline derivatives allowed three parallel bioorthogonal reactions to be conducted in one solution.
In Fmoc solid-phase peptide synthesis, the peptide chain is assembled stepwise, one amino acid at a time, while attached to an insoluble resin support. This allows the reaction by-products to be removed at each step by simple washing. Amino acids are protected at their amino terminus by the Fmoc (9-fluorenylmethoxycarbonyl) group and coupled to the growing chain after activation of the carboxylic acid terminus. The Fmoc group is then removed by piperidine treatment and the process repeated. After the peptide has been assembled it is removed from the resin by treatment with trifluoroacetic acid (TFA). At the same time, protecting groups on amino acid side chains are also removed yielding the crude linear peptide. One-step purification by reverse-phase HPLC is often sufficient to obtain the peptide in >95% purity.
(Fmoc Solid Phase Peptide Synthesis – A Practical Approach
Synthetic peptides are increasingly important in biochemical, pharmacological, neurobiological, enzymological and molecular biological research. Use of Fmoc as temporary amino‐protecting group allows solid‐phase peptide synthesis with a milder acid cleavage process. Other advances in the ligation approach provide further potential for peptide synthesis.
Fmoc Solid-Phase Peptide Synthesis: A Practical Approach
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