We report here a method for the determination of the pKa of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and trityl-protected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pKa of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pKa of interest.

中文翻译：

---

用于测定 pH 响应水凝胶中组氨酸 pKa 的氘增强拉曼光谱

我们在此报告了一种在复杂或异质系统中测定组氨酸 pKa 的方法，该系统既不适合固态也适用于溶液 NMR 光谱。小心合成芴甲氧羰基和三苯甲基保护的 C2-氘化组氨酸，产生一种配备振动探针的氨基酸，可以很容易地将其掺入任何可通过标准固相方法获得的肽中。这种 C2-D 探针独特的拉曼活性伸缩振动的频率是组氨酸质子化状态的明确报告者。我们在这里研究了一种 pH 敏感肽，它在中性 pH 下自组装形成水凝胶。由于凝胶的超分子、柔软性质，无法通过 NMR 光谱研究肽中单独组氨酸残基的 pKa，这可能是造成这种 pH 依赖性行为的原因。