Design and Application of a Novel Chalcone Derivative Fluorescent Probe for Aminopeptidase N

doi:10.6023/cjoc202312029

Abstract

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)

Cite this article

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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Fig. & Tab. 8

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

Scheme 2. Synthesis route of the probe Ala-OMN

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)

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)

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)

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 )

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

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

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