BNPS-skatole

A very mild brominating agent has been introduced recently for modification of the tryptophan residue in proteins 119,289). On treatment of 2-(2-nitrophenylsulfenyl)-3-methyl-indole (NPS-skatole) (112) with one equiv. of NBS in 80% acetic acid, the positive bromide-containing compound 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindole-nine (BNPS-skatole) (113) was obtained. 

BNPS-skatole is a much more selective agent than NBS. After exposure of an amino acid mixture to 10 equiv. of BNPS-skatole in 50% acetic acid for 30 min at room temperature, tryptophan was completely absent, methionine was converted to methionine sulfoxide, and the other amino acids were recovered quantitatively. By contrast, if the amino acid mixture is oxidized with NBS, not only tryptophan, but also methionine, tyrosine, histidine, and cystine were completely absent. Cysteine, as expected, is easily oxidized by BNPS-skatole to 

As anticipated from the action of NBS on tryptophan peptides (295), BNPS-skatole was proved useful also for selective cleavage of tryptophanyl peptide bonds (289). With a variety of tryptophan containing peptides, 10-fold excess of reagent in 20 hours gave cleavage yields (30-59%) comparable to those obtained with NBS. However, BNPS-skatole is a selective reagent and does not cleave at sites other than tryptophanyl peptide bonds (119, 289). 

Since cleavage is time-dependent and since tryptophan reacts rapidly with the reagent, modification without any significant cleavage was checked when the reaction was carried out with a low excess of reagent and for shorter times. This was clearly proved with staphylococcal nuclease (289). Reaction with BNPS-skatole and subsequent reduction of the 4 partially oxidized methionine residues produced a derivative of nuclease with modification only of residue 140, the single tryptophan unit. 

When nuclease was incubated with 100 equiv. of BNPS-skatole in 50% acetic acid for 28 hours, cleavage at the tryptophanyl peptide bond occurred in addition to modification. The released COOH-terminal nonapeptide was identified as a single spot on paper electrophoresis and from the amino acid recovery on the analyzer. The yield of cleavage product was estimated as approximately 15%. No additional peptides were released by cleavage at any other site in the sequence on the basis of analysis by electrophoresis. 

---

Chemical cleavage at selective amino acid residues is an alternative method for scission of peptide bonds in target molecules. Cleavage at the Met-Xaa bond is achieved by CNBr, at the Trp-Xaa bond by the tryptophan-directed reagent 3-bromo-3-methyl-2-[(2-nitro-phenyl)sulfanyl]-3//-indole (BNPS-skatole) and at the Asp-Xaa bond by 2% formic acid.123 24 All these reactions are carried out at acidic pH under standard conditions for each reagent. 

BNPS-Skatole, 3-bromo-3-rnethyl-2-(2-nitrophenylmercapto)-3H-mdole NTCB, 2-Nitro-5-thiocyanobenzoic acid... 

Fontana A. Modification of tryptophan with BNPS-skatole (2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine). Methods Enzymol. 1972 25 419-423. 

Recently, 2-(2-nitrophenylsulfenyI)-3-methyl-3-bromoindolenine (BNPS-skatole) has replaced N-bromosuccinimide, which had been used frequently in earlier studies. At low reagent to protein tryptophan ratios, in 50% aqueous acetic acid, BNPS-skatole reacts selectively with tryptophan residues converting these to the oxindole derivative. Methionine is concommitantly converted to the sulfoxide. At high concentrations of reagent, slow selective cleavage (to the extent of 15-60%) of the peptide bonds involving tryptophanyl residues is... 

Efficient automatic and manual sequencing of proteins is dependent entirely on the availability of specific cleavage methods. A new superior high-yield method for the fragmentation of proteins on the C-terminal side of tryptophan has been described by Mahoney and Hermodson. lodosobenzoate is used in the presence of 4-cresol and the method should replace the less efficient 2-(2-nitrophenyl-sulphenyl)-3-methyl-3 -bromoindolenine (BNPS-skatole) and dimethylsulphoxide-HBr methods. The specificity of trypsin (E.C. 3.4.21.4) can be broadened by conversion of aspartyl residues to the carboxyamidomethylamine derivative and a new L-proline-specific endopeptidase has been described. ... 

Taking this approach a step further, Vestling and Fenselau digested bovine adenosine deaminase with BNPS-skatole, which cleaves at tryptophan residues and converts the terminal tryptophan to an oxolactone ... 

FIGURE 10.11 Electropherogram detected by scanning laser desorption of proteins produced by the reaction of BNPS-skatole and bovine adenosine deaminase, separated on polyacrylamide and electroblotted onto a PVDF membrane. Reprinted with permission from reference 17. 

The use of BNPS-skatole for the selective cleavage of the tryptophanyl peptide bond has been proved useful in sequence studies. The cleavage yields so far obtained (about 50-70%) with peptides (289, 371) and proteins (119) indicate that the reagent is the best so far available for the cleavage of the tryptophanyl peptide bond and approaches in utility the cyanogen bromide cleavage of methionine peptide bonds (375). The reagent has been successfully applied to staphylococcal nuclease... 

In comparing NBS, TBC, N-bromo-benzotriazole and BNPS-skatole, it appears that the latter may offer certain advantages. BNPS-skatole is much less destructive toward tyrosine, histidine and cystine than the others, and this may be an important factor in choosing between them for a particular purpose. In addition, by exposing a tryptophan-containing protein to low amounts of BNPS-skatole and for a short time, it is possible to obtain modification and not cleavage of the tryptophanyl peptide bond. 

The use of BNPS-skatole (113) for the selective chemical cleavage of tryptophanyl peptide bonds has been further reported 459-462, 467). The selectivity of the reagent and the good yields of peptide bond fission clearly indicate that BNPS-skatole is of general utility for fragmentation of polypeptides and proteins, especially for sequence analysis. 

Fontana, A. Modification of Tryptophan with BNPS-Skatole (2-(2-Nitrophenyl-sulfenyl)-3-Methyl-3-Bromoindolenine), in Enzyme Structure. Methods in Enzymology (C. H. W. Hirs and S. N. Timasheff, Eds.). 25, 419-423. New York Academic Press. 1972. 

---