Pyridine borane as a reducing agent for proteins

Abstract

Pyridine borane has been reported as a superior reagent over a wide pH range, 5–9, for the reductive methylation of amino groups of proteins with formaldehyde [ J. C. Cabacungan, A. I. Ahmed, and R. E. Feeney (1982) Anal. Biochem., 124, 272–278]. It has also been reported to reduce tryptophan to dihydrotryptophan and to inactivate lysozyme in trifluoroacetic acid [ M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull., 28, 2274–2275]. In the present study the specificity of pyridine borane for the two different modifications under different reaction conditions has been demonstrated, and extended to the application to the synthesis of protein containing reductively attached carbohydrates. In the acid reduction, pyridine borane selectively reduced all six tryptophans in lysozyme to dihydrotryptophan while all other amino acids remained intact. On similar treatment no cleavage of the carbohydrate moiety from chicken ovomucoid, and no losses of activity of ovomucoid or ribonuclease, two proteins devoid of tryptophan, were observed. Nearly complete methylation of the lysines of lysozyme, chicken ovomucoid, and ribonuclease was achieved with formaldehyde at pH 7.0 after 2 h at room temperature, with the retention of full activity of the protein without any destruction of tryptophan. The same chemistry was applied to covalently attach glucose and lactose to bovine serum albumin. Parameters, including pH, temperature, and methanol, that effect the reactions were investigated. Incremental additions of pyridine borane during the course of the reaction increased the rate of modification. The covalent attachment of sugar to the ϵ-amino group of lysine was demonstrated by the synthesis of N-α-acetylglucitollysine and comparison with acid hydrolysates of the bovine serum albumin-sugar derivatives.