9‐Fluorenylmethyloxycarbonyl‐basierte Festphasensynthese von α‐Peptidthioestern

Mende, Franziska; Seitz, Oliver

doi:10.1002/ange.201005180

Cited by 37 publications

(15 citation statements)

References 93 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: 71%

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: 71%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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“…This approach avoids the alternative 9-fluorenyloxycarbonyl (Fmoc) SPPS based methods, which require a significant number of additional steps [18] to install the thioester, which is unstable towards the standard Fmoc deblocking reagent, piperidine. [19] In addition, fragments 2 and 5 both contain a Cterminal proline residue, which is known to undergo diketopiperazine [20] formation during Fmoc SPPS, [21] resulting in the final polypeptide lacking the first two amino acids. This undesired side reaction can be minimised when using Boc-based solid-phase methods.…”

Section: Synthesis Of Peptide Building Blocksmentioning

confidence: 99%

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Harris

Yang

Molina

et al. 2014

Chemistry A European J

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Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.

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“…The first step (Scheme 1, ligation 1) is an MPAA-catalyzed NCL between the alkylthioester group [16] of segment A and the Cys residue of the H-Cys-B-SEA off segment, leading to the formation of A-Cys-B-SEA off . The internal segment H-Cys-B-SEA off is a key element in this strategy.…”

mentioning

confidence: 99%

A One‐Pot Three‐Segment Ligation Strategy for Protein Chemical Synthesis

Ollivier¹,

Vicogne²,

Vallin³

et al. 2011

Angew Chem Int Ed

128

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Three in one: Native chemical ligation (NCL) and bis(2‐sulfanylethyl)amido (SEA) ligation allow the one‐pot assembly of three peptide segments in the N‐to‐C direction. The SEA group (see picture, blue) is switched off by intramolecular disulfide bond formation during NCL. Then, a phosphine switches it on to trigger the second SEA ligation step. The K1 domain of the hepatocyte growth factor was synthesized and found to be biologically active.

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9‐Fluorenylmethyloxycarbonyl‐basierte Festphasensynthese von α‐Peptidthioestern

Cited by 37 publications

References 93 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

A One‐Pot Three‐Segment Ligation Strategy for Protein Chemical Synthesis

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