New method of linking tryptophan to cysteine sulphydryl groups in peptides and proteins

Savige, W. E.; Fontana, Angelo

doi:10.1039/c39760000600

Cited by 47 publications

(27 citation statements)

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“…[24,25] This strategy has previously been used to good effect in the total synthesis of analogues of both amatoxins [26,27] and phallotoxins. [22] The requisite Hpi heptapeptide precursors for cyclization (3 a ± 3 h), bearing acid-labile protecting groups, were synthesized by the classical solution method.…”

Section: Synthesismentioning

confidence: 99%

Phalloidin Synthetic Analogues: Structural Requirements in the Interaction with F-Actin

Falcigno¹,

Costantini²,

D’Auria³

et al. 2001

Chem. Eur. J.

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Synthetic derivatives of phalloidin have been investigated in solution by circular dichroism (CD) and NMR spectroscopy. They differ from natural phalloidin (PHD), bicyclo(Ala 1 -d-Thr 2 -Cys 3 -cis-4-hydroxy-Pro 4 -Ala 5 -2-mercapto-Trp 6 -(OH) 2 Leu 7 )(S-3 3 6), in that they are modified at positions 2, 3, and 7. Among these synthetic analogues, structural differences and varying degrees of atropisomerism are found. By comparing the respective molecular models obtained by restrained molecular dynamics (RMD) simulations based on experimental NMR data, structural features that may be responsible for the different biological behavior become apparent. Our results indicate that the structural changes that result from an inversion of chirality of residue 3 lead to a complete loss of toxicity. Conversely, toxicity is less affected by the structural changes that stem from an inversion of chirality of residue 2. Moreover, unlike the other phallotoxins, when the thioether unit bridges to the opposite face of the main peptide ring, in contrast to the situation in other phallotoxins, large structural changes are observed as well as a total loss of activity. Molecular models of the synthetic phalloidin analogues have been used to investigate the necessary structural requirements for the interaction with F-actin. To this end, the F-actin/PHD model of M. Lorenz et al. was employed; docking experiments of our molecular models in the PHD binding site are presented.

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Section: Synthesismentioning

confidence: 99%

Phalloidin Synthetic Analogues: Structural Requirements in the Interaction with F-Actin

Falcigno¹,

Costantini²,

D’Auria³

et al. 2001

Chem. Eur. J.

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“…The procedure for the transformation of the respective linear octapeptides into the amanitin bicyclic structure was based on the “Savige Fontana” technique [28]. The monocyclic synthesis precursors HDP30.0445 and HDP30.0528 (Figure 1D) contain an open left side.…”

Section: Methodsmentioning

confidence: 99%

A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

et al. 2014

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BackgroundMany studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative.ResultsHerein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency.ConclusionsOur novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.

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“…In this paper, we describe a procedure for obtaining Oia-derivatives by treating a solution of tryptophan containing peptides in acetic acid with a mixture of DMSO and 12 N HCl. Preliminary reports of parts of this study have appeared (Savige & Fontana, 1976a, 1977a.…”

Section: **mentioning

confidence: 99%

ChemInform Abstract: OXIDATION OF TRYPTOPHAN TO OXINDOLYLALANINE BY DIMETHYL SULFOXIDE‐HYDROCHLORIC ACID. SELECTIVE MODIFICATION OF TRYPTOPHAN CONTAINING PEPTIDES

Savige¹,

Fontana²

1980

Chemischer Informationsdienst

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Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCI at room temperature. Other sulfoxides can be used in com bination with HCI; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of m eth ion in e and oxin do ly lalan in e. Me th ion ine and cys rein e are q uan tita tively oxidized by DMSOjHCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides L R F (luteinizing hormone-releusing factor), somatostatin, valine-gramicidin A and ACTU i-24 were each treated with the LIMSU/HCl reagent in acetic acid solution and the corresponding oxindobilalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis o f uhraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content o f acid hydrolyzates o f the oxidized peptides with 3N-p-toluenesulfonic acid at 11 0" for 24 h.The results indicate that modification of tryptophan peptides with DMSOIHCI provides a upe ful procedure, which seems superior to previously used reagents.In addition, the method could be well applied to other indoles of biological and pharmacological interest.brornosuccinirnide; BNPS-skatole, 2-(2-nitrophenyl-su~fenyI)-3-bromo-3-methytindolenine; RNase, bovine pancreatic ribonuclease A; t.1.c. thin-layer chromatozraphy. Amino acids and amino acid derivatives have the Li'onfiguration unless otherwise stated.

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New method of linking tryptophan to cysteine sulphydryl groups in peptides and proteins

Cited by 47 publications

References 0 publications

Phalloidin Synthetic Analogues: Structural Requirements in the Interaction with F-Actin

Phalloidin Synthetic Analogues: Structural Requirements in the Interaction with F-Actin

A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

ChemInform Abstract: OXIDATION OF TRYPTOPHAN TO OXINDOLYLALANINE BY DIMETHYL SULFOXIDE‐HYDROCHLORIC ACID. SELECTIVE MODIFICATION OF TRYPTOPHAN CONTAINING PEPTIDES

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