新型查尔酮衍生物氨肽酶N荧光探针的设计及应用

doi:10.6023/cjoc202312029

摘要/Abstract

氨肽酶N (APN)是一种广泛存在于生物体内的水解酶, 主要负责水解蛋白质多肽链N端的中性或碱性氨基酸. APN的过量表达与肿瘤、药物性肝损伤、急性肾损伤等多种生物功能失调密切相关, 被广泛应用于相关疾病的早期诊断与治疗. 尤其在肾小球肾炎的发生过程中, 尿液中APN含量的变化(升高)更早于其他蛋白水平的异常, 因而成为早期肾损伤的关键标志物之一. 基于4-氨基查尔酮结构设计合成了染料分子Ala-OMN. Ala-OMN由于4位氨基被丙氨酸笼蔽而没有荧光发射. 在APN存在的情况下, 丙氨酸基团被水解释放出OMN. 水解产生的OMN可以与磷酸盐缓冲溶液(PBS)中存在的牛血清白蛋白(BSA)形成具有强烈绿色荧光发射的复合物, 从而实现对APN的荧光响应. 实验结果表明Ala-OMN对APN的响应具有优异的灵敏度、选择性和抗干扰性, 检测限低至0.058 ng/mL. 在体外实验的基础上, 探针Ala-OMN成功实现了对尿液样本中APN水平的荧光检测, 为肾小球肾炎等肾脏疾病的早期诊断提供了潜在试剂.

关键词: 查尔酮衍生物, 氨肽酶N, 荧光探针, 牛血清白蛋白

Aminopeptidase N (APN) is a widely expressed hydrolytic enzyme in organisms that can hydrolyze neutral or alkaline amino acids at the N-terminus of protein polypeptide chains. Overexpressed APN may indicate disorders of biosystems, such as various tumors, drug-induced liver injury (DILI), and glomerulonephritis, and is widely used for early diagnosis and therapy of related diseases. Particularly, elevated APN level in urine is becoming one of the key markers of early kidney injury due to its special sensitivity to glomerulonephritis than other proteins. Herein, based on the structure of 4-amino chalcone, a novel fluorescent probe Ala-OMN for detecting APN was designed and synthesized by caging the 4-amino of OMN by alanine. Ala-OMN is non-fluorescent in phosphate buffered saline (PBS). After reaction with APN, the caging group alanine was hydrolyzed and OMN was released, which further combined with bovine serum protein (BSA) to form a 1∶1 complex, concomitantly with a bright green fluorescence signal for indicating APN. Ala-OMN exhibits excellent sensitivity, selectivity, and anti-interference ability in response to APN, with a detection limit as low as 0.058 ng/mL. Ala-OMN has been successfully applied to detect APN concentration in urine samples, and the results are in good agreement with commercial reagent kits, suggesting that Ala-OMN could be a new diagnostic reagent for the clinical detection of early glomerulonephritis.

Key words: chalcone derivatives, aminopeptidase N (APN), fluorescent probe, bovine serum protein (BSA)

引用此文

胡亦然, 张巳亮, 罗海艳, 赵璐瑶, 郭旭东, 王双青, 胡睿, 杨国强. 新型查尔酮衍生物氨肽酶N荧光探针的设计及应用[J]. 有机化学, 2024, 44(7): 2257-2264.

Yiran Hu, Siliang Zhang, Haiyan Luo, Luyao Zhao, Xudong Guo, Shuangqing Wang, Rui Hu, Guoqiang Yang. Design and Application of a Novel Chalcone Derivative Fluorescent Probe for Aminopeptidase N[J]. Chinese Journal of Organic Chemistry, 2024, 44(7): 2257-2264.

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图/表 8

图式1. 探针Ala-OMN与APN反应荧光点亮的机理示意图

Scheme 1. Fluorescence turn-on mechanism of the probe Ala-OMN to APN

图式2. 探针Ala-OMN的合成路线

Scheme 2. Synthesis route of the probe Ala-OMN

图1. OMN (10 μmol/L)和Ala-OMN (10 μmol/L)在PBS缓冲溶液[pH=7.4, 含DMSO (φ=1%)]及含1 mg/mL BSA的PBS缓冲溶液[pH=7.4, 含DMSO (φ=1%)]中的(A)吸收光谱和(B)荧光光谱(λex=420 nm)

Figure 1. (A) Absorpsion, and (B) fluorescence spectra of OMN (10 μmol/L) and Ala-OMN (10 μmol/L) in PBS buffer [pH=7.4, DMSO (φ=1%)], and PBS buffer with 1 mg/mL BSA [pH=7.4, DMSO (φ=1%)] (λex=420 nm)

图2. (A) Ala-OMN (10 μmol/L)与APN (50 ng/mL)在PBS溶液及含1 mg/mL BSA的PBS溶液中共孵育2 h (37 ℃)后的荧光光谱(λex=420 nm); (B) Ala-OMN (10 μmol/L)和APN (10 ng/mL)的混合溶液在孵育不同时间点时加入等量BSA后512 nm处荧光随时间的变化(λex=420 nm, 箭头指示了加入BSA的时间点)

Figure 2. (A) Fluorescence spectra of Ala-OMN (10 μmol/L) in PBS buffer and PBS buffer with 1 mg/mL BSA respectively treated with and without APN (50 ng/mL, 37 ℃, 2 h) (λex=420 nm); (B) Time dependent manner of the fluorescence intensity at 512 nm of the mixture of Ala-OMN (10 μmol/L) and APN (10 ng/mL) in PBS with addition of BSA at different incubation time (λex=420 nm. Arrows represent the BSA addition time)

图3. (A) Ala-OMN (10 μmol/L)对不同浓度APN (0~50 ng/mL, 含1 mg/mL BSA的PBS缓冲溶液, 37 ℃, 2 h)的荧光光谱响应(λex=420 nm); (B) A图中512 nm处的荧光强度与APN浓度(0~10 ng/mL)关系及线性拟合结果(n=3)

Figure 3. (A) Fluorescence spectra of Ala-OMN (10 μmol/L) with various concentration of APN (0~50 ng/mL, PBS buffer with 1 mg/mL BSA, 37 ℃, 2 h) (λex=420 nm); (B) Linear relationship of the fluorescence intensity at 512 nm in (A) with the concentration of APN (0~10 ng/mL) (n=3)

图4. (A)乌苯美司(20 μmol/L)预先处理及未处理的Ala-OMN (10 μmol/L)对APN (50 ng/mL)的荧光光谱响应(含1 mg/mL BSA的PBS缓冲溶液, 37 ℃, 2 h) (λex=420 nm); (B) Ala-OMN (100 μmol/L)与APN (50 ng/mL)反应1 h后的HR-ESI测试图(去离子水, 37 ℃)

Figure 4. (A) Fluorescence spectra of the mixture of Ala-OMN (10 μmol/L) and APN (50 ng/mL) pretreated with and without ubenimex (20 μmol/L) (PBS buffer with 1 mg/mL BSA, 2 h); (B) HR-ESI of the mixture of Ala-OMN (100 μmol/L) and APN (50 ng/mL) (deionized water, 37 ℃, 1 h )

图5. Ala-OMN (10 μmol/L)对APN (50 ng/mL)的选择性荧光响应(在含1 mg/mL BSA的PBS缓冲溶液中, 37 ℃, 2 h): (A) Ala-OMN与APN及其他干扰物分别孵育2 h后的荧光信号; (B) Ala-OMN与APN的共混体系在分别加入各种干扰物孵育后的荧光信号

Figure 5. Selective fluorescence response of Ala-OMN (10 μmol/L, in PBS buffer with 1 mg/mL BSA, 37 ℃, 2 h) to APN (50 ng/mL): (A) the signal intensity of Ala-OMN incubated with APN and other interferants respectively; (B) the signal intensity of the mixture of Ala-OMN and APN with other interferants (0) Blank, (1) Na+, (2) K+, (3) Fe3+, (4) Mg2+, (5) Cl–, (6) Br–, (7) I–, (8) $\mathrm{SO}_4^{2-}$, (9) Cys, (10) Hcy, (11) GSH, (12) glucose, (13) glycine, (14) alanine, (15) NaClO, (16) GGT, (17) ALP, (18) carboxylesterases 2, (19) Ache, (20) APN; n=3.

分组	Ala-OMN检测结果/(ng•mL^–1)	商用试剂盒检测/(ng•mL^–1)	回收率/%
正常人尿液	436±13	454±39	96
正常人尿液+500 ng/mL APN	958±17	937±34	102
正常人尿液+1000 ng/mL APN	1426±28	1389±78	103
正常人尿液+1500 ng/mL APN	1947±58	1889±57	103

表1. Ala-OMN和APN商用试剂盒对人尿液环境中APN的检测结果a

Table 1. Detection results of Ala-OMN and commercial APN ELISA for APN in human urine

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