Wong W S, Osuga D T, Feeney R E
Anal Biochem. 1984 May 15;139(1):58-67. doi: 10.1016/0003-2697(84)90388-9.
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 affect the reactions were investigated. Incremental additions of pyridine borane during the course of the reactions increased the rate of modification. The covalent attachment of sugar to the epsilon-amino group of lysine was demonstrated by the synthesis of N-alpha- acetylglucitollysine and comparison with acid hydrolysates of the bovine serum albumin-sugar derivatives.
据报道，在5 - 9的广泛pH范围内，吡啶硼烷是一种比甲醛更优越的试剂，可用于蛋白质氨基的还原甲基化反应[J. C. 卡巴昆甘、A. I. 艾哈迈德和R. E. 费尼（1982年），《分析生物化学》124卷，272 - 278页]。也有报道称，它能将色氨酸还原为二氢色氨酸，并在三氟乙酸中使溶菌酶失活[仓田真、菊川义之、桑江彻、小山一和高木敏郎（1980年），《化学与药学通报》28卷，2274 - 2275页]。在本研究中，已证明吡啶硼烷在不同反应条件下对这两种不同修饰的特异性，并将其扩展应用于含还原性连接碳水化合物的蛋白质合成。在酸性还原反应中，吡啶硼烷能选择性地将溶菌酶中的所有六个色氨酸还原为二氢色氨酸，而所有其他氨基酸保持完整。经过类似处理后，未观察到鸡卵类粘蛋白的碳水化合物部分被裂解，也未观察到卵类粘蛋白或核糖核酸酶（两种不含色氨酸的蛋白质）的活性丧失。在室温下，用甲醛在pH 7.0条件下处理2小时后，溶菌酶、鸡卵类粘蛋白和核糖核酸酶的赖氨酸几乎完全甲基化，同时蛋白质保留了全部活性，色氨酸未被破坏。相同的化学方法被用于将葡萄糖和乳糖共价连接到牛血清白蛋白上。研究了影响反应的参数，包括pH、温度和甲醇。在反应过程中逐步添加吡啶硼烷可提高修饰速率。通过合成N-α-乙酰葡糖胺赖氨酸并与牛血清白蛋白-糖衍生物的酸水解产物进行比较，证明了糖与赖氨酸的ε-氨基共价连接。
