Bailey T Spencer, Pluth Michael D
J Am Chem Soc. 2013 Nov 6;135(44):16697-704. doi: 10.1021/ja408909h.
Hydrogen sulfide (H2S) is now recognized as an important biological regulator and signaling agent that is active in many physiological processes and diseases. Understanding the important roles of this emerging signaling molecule has remained challenging, in part due to the limited methods available for detecting endogenous H2S. Here we report two reaction-based ChemiLuminescent Sulfide Sensors, CLSS-1 and CLSS-2, with strong luminescence responses toward H2S (128- and 48-fold, respectively) and H2S detection limits (0.7 ± 0.3, 4.6 ± 2.0 μM, respectively) compatible with biological H2S levels. CLSS-2 is highly selective for H2S over other reactive sulfur, nitrogen, and oxygen species (RSONS) including GSH, Cys, Hcy, S2O3(2–), NO2(–), HNO, ONOO(–), and NO. Despite its similar chemical structure, CLSS-1 displays lower selectivity toward amino acid-derived thiols than CLSS-2. The origin of this differential selectivity was investigated using both computational DFT studies and NMR experiments. Our results suggest a model in which amino acid binding to the hydrazide moiety of the luminol-derived probes provides differential access to the reactive azide in CLSS-1 and CLSS-2, thus eroding the selectivity of CLSS-1 for H2S over Cys and GSH. On the basis of its high selectivity for H2S, we used CLSS-2 to detect enzymatically produced H2S from isolated cystathionine γ-lyase (CSE) enzymes (p < 0.001) and also from C6 cells expressing CSE (p < 0.001). CLSS-2 can readily differentiate between H2S production in active CSE and CSE inhibited with β-cyanoalanine (BCA) in both isolated CSE enzymes (p < 0.005) and in C6 cells (p < 0.005). In addition to providing a highly sensitive and selective reaction-based tool for chemiluminescent H2S detection and quantification, the insights into substrate–probe interactions controlling the selectivity for H2S over biologically relevant thiols may guide the design of other selective H2S detection scaffolds.
硫化氢（H₂S）现在被认为是一种重要的生物调节剂和信号分子，在许多生理过程和疾病中发挥作用。了解这种新兴信号分子的重要作用仍然具有挑战性，部分原因是检测内源性H₂S的可用方法有限。在此，我们报告了两种基于反应的化学发光硫化物传感器CLSS - 1和CLSS - 2，它们对H₂S具有强烈的发光响应（分别为128倍和48倍），并且H₂S检测限（分别为0.7±0.3、4.6±2.0 μM）与生物体内H₂S水平相匹配。CLSS - 2对H₂S的选择性高于其他活性硫、氮和氧物种（RSONS），包括谷胱甘肽（GSH）、半胱氨酸（Cys）、同型半胱氨酸（Hcy）、硫代硫酸根离子（S₂O₃²⁻）、亚硝酸根离子（NO₂⁻）、亚硝酸（HNO）、过氧亚硝酸根离子（ONOO⁻）和一氧化氮（NO）。尽管CLSS - 1和CLSS - 2化学结构相似，但CLSS - 1对氨基酸衍生的硫醇的选择性低于CLSS - 2。使用计算密度泛函理论（DFT）研究和核磁共振（NMR）实验对这种选择性差异的来源进行了研究。我们的结果提出了一个模型，其中氨基酸与鲁米诺衍生探针的酰肼部分结合，为CLSS - 1和CLSS - 2中的活性叠氮化物提供了不同的可及性，从而削弱了CLSS - 1对H₂S相对于Cys和GSH的选择性。基于其对H₂S的高选择性，我们使用CLSS - 2检测了从分离的胱硫醚γ - 裂解酶（CSE）酶（p < 0.001）以及表达CSE的C6细胞中酶促产生的H₂S（p < 0.001）。在分离的CSE酶（p < 0.005）和C6细胞（p < 0.005）中，CLSS - 2能够轻松区分活性CSE产生的H₂S和被β - 氰基丙氨酸（BCA）抑制的CSE产生的H₂S。除了为化学发光H₂S检测和定量提供一种高度灵敏且选择性的基于反应的工具外，对控制H₂S相对于生物相关硫醇选择性的底物 - 探针相互作用的深入了解可能会指导其他选择性H₂S检测支架的设计。
