荧光素酶-腔肠素 (CTZ) 反应产生的生物发光 (BL) 广泛用作生物测定和体内分子成像中的光学读数。在这项研究中，我们展示了一种系统方法，用一整套区域异构 CTZ 类似物来阐明荧光素酶-CTZ 结合化学，其中所有官能团都经过区域化学修饰。当化学结构分为第 1-6 组时，发现 CTZ 类似物的偶数组（2、4 和 6）在 NanoLuc 酶的作用下异常明亮。 CTZ 类似物 M2 在 NanoLuc 中最亮，其原因通过结合模式的计算分析得以解释。我们还报道，（i）区域异构CTZ类似物根据每种海洋荧光素酶共同创建独特的强度模式，（ii）定量结构-活性关系分析揭示了CTZ类似物各自功能组的作用，以及（iii）区域异构CTZ类似物也会产生 BL 光谱的红移和颜色变化：也就是说，NanoLuc 的 λmax 值接近 500 nm，ALuc16 的 λmax 值接近 530 nm，RLuc86SG 的 λmax 值接近 570 nm。最终使用（i）具有M2特异性NanoLuc和天然CTZ特异性ALuc16的双荧光素酶系统，（ii）通过成像的雌激素可激活单链BL探针，以及（iii）BL证明了区域异构CTZ类似物的优点对携带表达 NanoLuc 和 RLuc8.6SG 的肿瘤的活小鼠进行成像。这项研究是阐明 BL 成像研究中区域化学的第一个系统方法。这项研究为 CTZ 类似物在 BL 报告系统中的区域化学作用提供了新的见解，并指导了分子成像的具体应用。

Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λmax values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2-specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.