1. Edman degradation sequence analysis of resin-bound peptides synthesized by 9-fluorenylmethoxycarbonyl chemistry
C G Fields, V L VanDrisse, G B Fields Pept Res. 1993 Jan-Feb;6(1):39-47.
The efficacy of Edman degradation sequence analysis for evaluating the synthetic efficiency of peptide-resin assembly by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase methodology has been studied. Prior researchers have described the use of solid-phase "preview" sequence analysis for peptides synthesized by tertiary-butyloxycarbonyl (Boc) chemistry, where benzyl-based side-chain protecting groups and peptide-resin linkers are stable to the conditions of Edman chemistry. We have successfully sequenced a variety of resin-bound peptides synthesized by Fmoc chemistry, where tertiary-butyl-based side-chain protecting groups and peptide-resin linkers are labile to the conditions of Edman chemistry. Crude peptides are liberated from trifluoroacetic acid-labile linkers during the first cycle of Edman degradation and subsequently "embedded" in membranes. For peptides up to 20 residues, embedded sequencing repetitive yields were comparable to those of solid-phase sequencing. Preview sequencing of resin-bound Fmoc-synthesized peptides proved to be advantageous compared to other analytical methods, in that synthetic failures were detected and quantitated at the point of occurrence, regardless of whether incomplete Fmoc deprotection or incomplete coupling was responsible, and without interference from by-products formed during peptide-resin cleavage. Quantitative ninhydrin analysis, which previously has been found to give false positive results due to removal of the Fmoc group by a combination of reagents and high temperature, gave false negative results in this study, most probably due to incomplete removal of the Fmoc group prior to coupling. Quantitative sequence analysis results were supported by high-performance liquid chromatographic, amino acid and electrospray mass spectrometric analyses of the crude and purified peptides.
2. Preparation and Use of a General Solid-Phase Intermediate to Biomimetic Scaffolds and Peptide Condensations
J Geno Samaritoni, Jacek G Martynow, Martin J O'Donnell, William L Scott Molecules. 2018 Jul 18;23(7):1762. doi: 10.3390/molecules23071762.
The Distributed Drug Discovery (D3) program develops simple, powerful, and reproducible procedures to enable the distributed synthesis of large numbers of potential drugs for neglected diseases. The synthetic protocols are solid-phase based and inspired by published work. One promising article reported that many biomimetic molecules based on diverse scaffolds with three or more sites of variable substitution can be synthesized in one or two steps from a common key aldehyde intermediate. This intermediate was prepared by the ozonolysis of a precursor functionalized at two variable sites, restricting their presence in the subsequently formed scaffolds to ozone compatible functional groups. To broaden the scope of the groups available at one of these variable sites, we developed a synthetic route to an alternative, orthogonally protected key intermediate that allows the incorporation of ozone sensitive groups after the ozonolysis step. The utility of this orthogonally protected intermediate is demonstrated in the synthesis of several representative biomimetic scaffolds containing ozonolytically labile functional groups. It is compatible with traditional Fmoc peptide chemistry, permitting it to incorporate peptide fragments for use in fragment condensations with peptides containing cysteine at the N-terminus. Overall yields for its synthesis and utilization (as many as 13 steps) indicate good conversions at each step.
3. Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?
Omar F Luna, Johana Gomez, Constanza Cárdenas, Fernando Albericio, Sergio H Marshall, Fanny Guzmán Molecules. 2016 Nov 15;21(11):1542. doi: 10.3390/molecules21111542.
The deprotection step is crucial in order to secure a good quality product in Fmoc solid phase peptide synthesis. 9-Fluorenylmethoxycarbonyl (Fmoc) removal is achieved by a two-step mechanism reaction favored by the use of cyclic secondary amines; however, the efficiency of the reaction could be affected by side reactions and by-product formation. Several aspects have to be taken into consideration when selecting a deprotection reagent: its physicochemical behavior, basicity (pKa) and polarity, concentration, and time of reaction, toxicity and disposability of residues and, finally, availability of reagents. This report presents a comparison of the performance of three strategies for deprotection using microwave-assisted Fmoc peptide synthesis. Four peptide sequences were synthesized using Rink amide resin with a Liberty Blue™ automated synthesizer and 4-methylpiperidine (4MP), piperidine (PP), and piperazine (PZ) as Fmoc removal reagents. In the first instance all three reagents behaved similarly. A detailed analysis showed a correlation between the hydrophobicity and size of the peptide with the yield and purity of the obtained product. The three reagents are interchangeable, and replacement of piperidine could be advantageous regarding toxicity and reagent handling.
