Practical Protein Chemistry - A. Darbre 1989

Chemical Fragmentation of Polypeptides
Cleavage at Tryptophan Residues
Cleavage Using BNPS-Skatole

Currently, 2-(2-nitrophenylsulfenyl)-3-bromo-3-methylindolenine (57) (BNPS-skatole) is primarily used for the Cleavage of Peptides and Proteins at Tryptophan residues [45, 50, 132]. The reagent is prepared by brominating 2-(2-nitrophenylsulfenyl)-3-methylindole (56) with N-Bromosuccinimide in glacial acetic acid [132]. A modified method for synthesizing BNPS-skatole was proposed in [211], where its "indolenine" Structure was also spectroscopically confirmed.

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BNPS-skatole acts much more selectively compared to N-bromosuccinimide. Treatment of an amino acid mixture with BNPS-skatole (10 equiv.) in 50% acetic acid at room Temperature for 30 min leads to the complete disappearance of tryptophan, The conversion of Methionine to methionine sulfoxide, while all Other Amino Acids are recovered quantitatively in the hydrolysate. Conversely, under identical treatment with N-bromosuccinimide, not only tryptophan is absent from the hydrolysate, but also methionine, Tyrosine, Histidine, and cystine. As expected, Cysteine is readily oxidized by BNPS-skatole to cystine, and partially to cysteic acid in the presence of an excess reagent. If SH-groups are present in the protein, they must be protected beforehand using a suitable reagent [132].

As convincingly demonstrated using nuclease as an example [132], Cleavage at Tryptophan Residues requires prolonged incubation in the presence of a reagent excess (20- to 100-fold). Shorter reaction times and a small excess of the reagent result in only partial modification of tryptophan.

Numerous studies describe the application of BNPS-skatole in Protein Chemistry [51]. The yield of products resulting from selective cleavage at tryptophan ranges from 40% to 80%. This yield apparently depends on the quality of the reagent. BNPS-skatole is light-sensitive, decomposes at room temperature, darkens, and releases bromine. At the same time, highly purified crystalline preparations (pale yellow rosette-like crystals obtained from ligroin crystallization [132]) can be stored in airtight containers at —20 °C for a sufficiently long time.

2.5.2.1. Procedure [123]. The peptide comprising residues 89–169 of bovine myelin basic protein (1–7 µmol/mL) is incubated with BNPS-skatole (17 µmol/mL) in 75% acetic acid at 37 °C for 24 h. Subsequently, the excess reagent and reaction byproducts are removed by repeated extraction with ethyl acetate, and the aqueous layer is lyophilized. The peptide fragments are purified using Gel filtration.