Peptide αthioester formation using standard Fmoc-chemistry

Eggelkraut‐Gottanka, Regula von; Klose, Annerose; Beck‐Sickinger, Annette G.; Beyermann, Michael

doi:10.1016/s0040-4039(03)00582-3

Cited by 91 publications

(64 citation statements)

References 17 publications

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“…However, several studies have reported the presence of just traces of racemization at Gln thioesters [38,39,[42][43][44][45]48] when using protocols similar to those used in our study (PyBOP-coupling conditions); this rules out racemization at the Gln residue of Rev3. On the other hand, the level of racemization in the case of the C-terminal Ser thioester, which we prepared by using BF 3 ·etherate on trichloroacetimidate activated Wang resin, has not been investigated.…”

Section: Chemical Synthesis Of Hiv-1 Revmentioning

confidence: 68%

“…The Rev3 thioester was prepared in similar yield by anchoring Fmoc-(Glu)-OAll on Rink amide resin (Supporting Information). [42][43][44][45] We then turned our focus to the synthesis of the full length Rev protein by applying the above-described strategy. We first ligated Rev1 to Rev2 under NCL conditions, followed by conversion of the 1,3-thiazolidine-4-carboxo (Thz) to Cys by using methoxylamine, [46] to give Rev2-Rev1 in 35 % isolated yield for the two steps ( Figure 1 A and B).…”

Section: Chemical Synthesis Of Hiv-1 Revmentioning

confidence: 99%

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Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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The HIV‐1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV‐1 lifecycle, thus making Rev an attractive target for the design of anti‐HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self‐assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one‐pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.

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Section: Chemical Synthesis Of Hiv-1 Revmentioning

confidence: 68%

Section: Chemical Synthesis Of Hiv-1 Revmentioning

confidence: 99%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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“…This approach was used to generate two different 20-mer peptide thioesters derived from the transmembrane S4 segment of the rabbit skeletal muscle calcium channel and from Alamethicin in moderate yield (16% to 20%). Since then, Futaki's approach has been successfully used by different groups for the synthesis of different Cholecystokinin (CCK) isoforms [28], N-terminal modified Histones [29], pro-neuropeptide Y fragments [30] and several polyglycosylated polypeptides [31]. In all cases the peptide thioester fragments involved in the chemical synthesis of the different proteins were generated by Fmoc-based SPPS on a 4-Cl-Trt resin with similar yields to those reported previously.…”

Section: Fmoc-based Synthesis Of Peptide A-thioestersmentioning

confidence: 81%

Synthesis of Proteins by Native Chemical Ligation Using Fmoc‐Based Chemistry

Camarero

Mitchell

2006

ChemInform

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“…The 14-amino acid long peptide Trp-Pro-PheLeu-Arg-His-Asn-Val-Tyr-Gly-Arg-Pro-Arg-Ala was prepared by solid-phase peptide synthesis and cleaved from the resin in protected form. Following activation of its C terminus as a thioester (19), side chain-protecting groups were removed, and the peptide was purified by RP-HPLC. The deprotected thioester reacted cleanly with selenocysteine under denaturing conditions in the presence of thiophenol to give 15SeP.…”

Section: Resultsmentioning

confidence: 99%

“…The recovered solid was resuspended in a small amount of dioxane, flash-frozen, and lyophilized. The crude protected peptide was dissolved in a 2:1 mixture of N,NЈ-dimethylformamide/dichloromethane (0.02 M) and converted to a thioester by adding PyBOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; 3 eq) in the presence of p-acetamidothiophenol (ArSH; 3 eq) and N,NЈ-diisopropylethylamine (3 eq) at 0°C (19). The mixture was allowed to warm to room temperature, and the solvent was evaporated after 3 h. A small amount of water was added to the crude mixture, which was then lyophilized.…”

Section: Methodsmentioning

confidence: 99%

Reinvestigation of a Selenopeptide with Purportedly High Glutathione Peroxidase Activity

Casi

Hilvert

2007

Journal of Biological Chemistry

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. Such a finding is startling considering the high efficiency of the natural enzyme and the modest catalytic properties of most short peptides. As 15SeP had been subjected only to limited chemical characterization, we prepared it by a new route involving selenocysteine-mediated native chemical ligation. High resolution matrix-assisted laser desorption ionization mass spectrometry confirmed the identity of the reaction product, whereas circular dichroism spectroscopy showed that 15SeP assumes a random coil conformation in solution. Although low levels of peroxidase activity were detectable under standard assay conditions, the peptide is >5 orders of magnitude less active than native Gpx. Our observations are incompatible with claims ascribing remarkable catalytic properties to 15SeP and suggest that the efficiency of Gpx derives from its well defined three-dimensional structure.

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Peptide αthioester formation using standard Fmoc-chemistry

Cited by 91 publications

References 17 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Synthesis of Proteins by Native Chemical Ligation Using Fmoc‐Based Chemistry

Reinvestigation of a Selenopeptide with Purportedly High Glutathione Peroxidase Activity

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