1,8-萘酰亚胺衍生物的设计、合成及其对半胱氨酸的识别研究

doi:10.6023/cjoc202306016

摘要/Abstract

巯基氨基酸水平异常与多种疾病相关, 其检测仍存在一定的局限, 研究检测巯基氨基酸的荧光探针具有一定的价值. 以苊为原料合成了61个1,8-萘酰亚胺衍生物, 研究了该类化合物的荧光性能及其作为半胱氨酸含量测定的荧光探针的可能性. 紫外光谱分析表明1,8-萘酰亚胺衍生物上N-取代基对最大吸收波长无明显影响; 荧光光谱(FL)的性能测试显示硝基萘酰亚胺衍生物N-甲基-4-硝基-1,8-萘二甲酰亚胺(4a)~4-硝基-N-间氟苯基-1,8-萘二甲酰亚胺(4s)无荧光, 氨基萘酰亚胺衍生物N-甲基-4-氨基-1,8-萘二甲酰亚胺(5a)~4-氨基-N-间氟苯基-1,8-萘二甲酰亚胺(5s)有强烈黄色荧光, 而马来酰亚胺萘酰亚胺衍生物N-甲基-4-(1H-吡咯-2,5-二酮-1-基)-1,8-萘二甲酰亚胺(6a)~4-(1H-吡咯-2,5-二酮-1-基)-N-(间氟苯基)-1,8-萘二甲酰亚胺(6s)有微弱蓝色荧光, 其中7个马来酰亚胺萘酰亚胺衍生物探针对半胱氨酸(Cys)溶液有荧光点亮效应. 对7个探针加入21种其它氨基酸作为干扰项的测试显示探针对半胱氨酸检测有良好的选择性. 研究了不同pH值下荧光强度, 检测探针与半胱氨酸的响应时间, 以及探针溶液荧光强度随氨基酸浓度的变化, 其线性相关系数均达到0.95以上, 探针对半胱氨酸的各项指标均表现出了较好的灵敏性. Hela细胞荧光成像验证了7个荧光探针应用于细胞内半胱氨酸的检测同样效果良好.

关键词: 合成, 荧光探针, 半胱氨酸, 细胞成像, 1,8-萘酰亚胺衍生物

Determination of sulfhydryl amino acid content is of great importance for disease research. Therefore, it is necessary to develop a fluorescent probe that can detect sulfhydryl amino acids. In this paper, sixty-one 1,8-naphthoyl derivatives were successfully synthesized. These performances of compounds were measured by UV-Vis spectra and fluorescence spectra (FL). Unlike the non-fluorescent nitro-substituted compounds N-methyl-4-nitro-1,8-napthalimide (4a)~N-(m-fluorophenyl)-4-nitro-1,8-napthalimide (4s), the amino-substituted compounds N-methyl-4-amino-1,8-napthalimide (5a)~N-(m-fluorophenyl)-4-amino-1,8-napthalimide (5s) have strong yellow fluorescence, and the maleic anhydride-sub- stituted compounds N-methyl-4-(1H-pyrrole-2,5-diketone-1-yl)-1,8-napthalimide (6a)~N-(m-fluorophenyl)-4-(1H-pyrrole- 2,5-diketone-1-yl)-1,8-napthalimide (6s) have weak blue fluorescence. Of the 61 probes, 7 probes have fluorescent OFF-ON effect on cysteine (Cys) solutions. It was found that these probes have the potential to detect cysteine. The specificity of the probe for cysteine detection was explored by adding 21 amino acids as distracters. The fluorescence intensity at different pH values were studied. The response time between the detection probe and cysteine, and the variation of the fluorescence intensity of the probe solution with amino acid concentration were also studied. The linear correlation coefficients reached above 0.95. The response time of the probe was tested by fluorescence time spectral scanning. Through the above experiments, it was found that the probe showed a better sensitivity and selectivity for cysteine. The ability of seven fluorescent probes to be used for intracellular cysteine detection was explored by Hela cell fluorescence imaging and screened out probes with potential for biological detection.

Key words: synthesis, fluorescence molecular probe, cysteine, 1,8-napthalimide derivatives, cell fluorescence imaging

引用此文

杨维清, 葛宴兵, 陈元元, 刘萍, 付海燕, 马梦林. 1,8-萘酰亚胺衍生物的设计、合成及其对半胱氨酸的识别研究[J]. 有机化学, 2024, 44(1): 180-194.

Weiqing Yang, Yanbing Ge, Yuanyuan Chen, Ping Liu, Haiyan Fu, Menglin Ma. Design and Synthesis of Fluorescent 1,8-Napthalimide Derivatives and Their Identification of Cysteine[J]. Chinese Journal of Organic Chemistry, 2024, 44(1): 180-194.

导出引用 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

分享此文

图/表 12

图1. 1,8-萘酰亚胺衍生物探针对半胱氨酸(Cys)选择性识别

Figure 1. 1,8-Napthalimide derivatives fluorescence probe and its identification of cysteine

图式1. 1,8-萘酰亚胺衍生物荧光探针的合成路线

Scheme 1. Synthesis of 1,8-napthalimide derivatives

Compd.	λ_max/nm	ε/(10³ L• mol^－¹•cm^－¹)	Compd.	λ_max/nm	ε/(10³ L• mol^－¹•cm^－¹)	Compd.	λ_max/nm	ε/(10³ L• mol^－¹•cm^－¹)	Compd.	λ_max/nm	ε/(10³ L• mol^－¹•cm^－¹)
4a	353	10.78	5a	436	13.60	6a	338	17.20	6a＋Cys	348	16.25
4b	352	10.86	5b	436	21.15	6b	346	16.10	6b＋Cys	366	9.60
4c	352	7.64	5c	434	11.40	6c	338	17.85	6c＋Cys	344	15.90
4d	350	11.29	5d	437	13.70	6d	353	9.00	6d＋Cys	351	7.45
4e	354	14.85	5e	441	10.15	6e	354	9.75	6e＋Cys	355	8.55
4f	352	13.70	5f	436	10.55	6f	352	7.10	6f＋Cys	351	5.70
4g	354	13.50	5g	437	8.10	6g	344	9.55	6g＋Cys	348	10.60
4h	355	12.60	5h	440	18.40	6h	346	7.30	6h＋Cys	344	8.20
4i	354	4.45	5i	434	10.85	6i	347	10.80	6i＋Cys	347	8.30
4j	354	8.90	5j	436	12.10	6j	344	12.25	6j＋Cys	349	13.30
4k	354	11.95	5k	437	9.65	6k	344	15.85	6k＋Cys	348	14.60
4l	356	12.70	5l	438	10.75	6l	348	13.20	6l＋Cys	349	12.10
4m	354	11.45	5m	437	8.80	6m	364	15.05	6m＋Cys	364	15.90
4n	353	13.45	5n	437	12.15	6n	348	14.25	6n＋Cys	354	14.20
4o	357	14.15	5o	438	15.15	6o	345	13.35	6o＋Cys	359	13.10
4p	357	13.80	5p	437	19.25	6p	345	12.40	6p＋Cys	345	11.15
4q	359	11.90	5q	437	17.10	6q	345	10.45	6q＋Cys	365	9.70
4r	352	8.75	5r	437	30.25	6r	342	12.15	6r＋Cys	359	10.55
4s	353	5.10	5s	436	7.55	6s	341	9.05	6s＋Cys	340	8.65

表1. 1,8-萘酰亚胺衍生物的紫外吸收性能

Table 1. UV absorption properties of 1,8-napthalimide derivatives

Compd.	λ_Ex/nm	λ_Em/nm	Hight^b	Compd.	λ_Ex/nm	λ_Em/nm	Hight^b	Compd.	λ_Ex/nm	λ_Em/nm	Φ_F^a/%
5a	440	537	214.2	6a	274	328	67.6	6a＋Cys	378	468	9.06
5b	440	537	200.7	6b	272	328	171.0	6b＋Cys	372	472	—^c
5c	276	327	167.0	6c	273	326	182.0	6c＋Cys	372	470	—^c
5d	440	536	213.5	6d	280	310	28.7	6d＋Cys	378	470	11.36
5e	440	537	191.8	6e	280	310	33.9	6e＋Cys	370	467	12.56
5f	440	538	149.6	6f	370	472	66.6	6f＋Cys	375	468	16.32
5g	270	327	75.62	6g	270	324	60.2	6g＋Cys	370	465	—^c
5h	257	340	209.0	6h	280	310	36.7	6h＋Cys	370	468	0.18
5i	440	539	83.35	6i	273	325	72.3	6i＋Cys	371	468	—^c
5j	438	540	135.7	6j	280	310	33.1	6j＋Cys	380	469	1.52
5k	440	538	119.6	6k	280	310	29.6	6k＋Cys	370	471	1.50
5l	440	536	167.8	6l	370	466	48.7	6l＋Cys	370	468	19.25
5m	442	540	97.3	6m	280	310	34.8	6m＋Cys	370	459	—^c
5n	440	538	85.3	6n	280	310	32.6	6n＋Cys	370	468	11.89
5o	280	311	36.3	6o	280	311	34.1	6o＋Cys	370	470	8.26
5p	440	542	105.9	6p	280	310	35.2	6p＋Cys	372	466	6.71
5q	440	540	161.4	6q	280	310	35.2	6q＋Cys	370	470	17.89
5r	440	538	98.6	6r	280	310	36.8	6r＋Cys	370	462	—^c
5s	440	542	35.0	6s	280	310	36.3	6s＋Cys	370	309	0.13

表2. 1,8-萘酰亚胺衍生物的荧光性能

Table 2. Fluorescence properties of 1,8-napthalimide derivatives

图2. 7种荧光探针在2 μmol/L的DMSO/HEPES (V∶V=1∶1)混合溶液中与22种氨基酸的荧光发射光谱

Figure 2. Fluorescence emission spectra of 7 fluorescent probes and 22 amino acids in 2 μmol/L DMSO/HEPES (V∶V=1∶1) solution

图3. 6q在2 μmol/L 的DMSO/HEPES (V∶V=1∶1)混合溶液中与三种氨基酸的作用结果图

Figure 3. Results of the interaction of 6q in 2 μmol/L DMSO/ HEPES (V∶V=1∶1) solution with three amino acids

图4. 在2 μmol/L 的DMSO/HEPES (V∶V=1∶11)混合溶液中470 nm波长下荧光探针的反应时间

Figure 4. Time-dependent fluorescence intensity of probe at 470 nm in 2 μmol/L DMSO/HEPES (V∶V=1∶1) solution

图5. 在2 μmol/L的DMSO/HEPES (V∶V=1∶1)的混合溶液中470 nm波长下pH对荧光强度的影响

Figure 5. Effect of pH on fluorescence intensity at 470 nm in 2 μmol/L DMSO/HEPES (V∶V=1∶1) solution

图6. Cys浓度从0到2.0 μmol?L－1递增探针浓度为20 μmol?L－1于470 nm处荧光强度影响

Figure 6. Effect of Cys concentration (0 to 2.0 μmol?L－1, from up to down) on fluorescence intensity of probe (20 μmol?L－1) at 470 nm

图7. Cys浓度(从上到下, 横坐标浓度从0到2.0 μmol?L－1)与探针(20 μmol?L－1)于470 nm处探针荧光强度的线性拟合

Figure 7. Linear fitting of Cys concentration (0 to 2.0 μmol?L－1, from up to down) on fluorescence intensity of probe (20 μmol?L－1) at 470 nm

图8. 5 μmol?L－1的6a, 6d, 6e, 6f, 6l, 6n和6q的细胞成像荧光图

Figure 8. Cell fluorescence diagrams of 5 μmol?L－1 6a, 6d, 6e, 6f, 6l, 6n and 6q (dark-field observation)

图9. 5 μmol?L－1的6a, 6d, 6e, 6f, 6l, 6n和6q的细胞成像荧光图(明场观察)

Figure 9. Cell fluorescence diagram of 5 μmol?L－1 6a, 6d, 6e, 6f, 6l, 6n and 6q (bright-field observation) (a) Bright-field cell diagram after adding probe 6a; (b) Dark-field cell diagram after adding probe 6a; (c) Bright-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6a; (d) Dark-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6a; (e) Bright-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6a and Cys; (f) Dark-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6a and Cys; (g) Bright-field cell diagram after adding probe 6d; (h) Dark-field cell diagram after adding probe 6g; (i) Bright-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6d; (j) Dark-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6d; (k) Bright-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6d and Cys; (l) Dark-field cell diagram of preprocessed with NEM before adding 5 μmol/L 6d and Cys.

参考文献 15

[1]	(a) Sun, Y.; Fu, M. L; Bian, M. L; Zhu, Q. Biotechnol Bioeng. 2023, 120, 7. doi: 10.1002/bit.v120.1
	(b) Ahmed, N.; Zareen, W.; Ye, Y. Chin. Chem. Lett. 2022, 33, 2765. doi: 10.1016/j.cclet.2021.12.092
	(c) Liu, F. R.; Jing, J.; Zhang, X. L. Chin. J. Org. Chem. 2023, 43, 2053 (in Chinese). doi: 10.6023/cjoc202209005
	(刘飞冉, 敬静, 张小玲, 有机化学, 2023, 43, 2053.)
[2]	(a) Kolinska, J.; Grzelakowska, A. Spectrochim. Acta, A Mol. Biomol. Spectrosc. 2021, 262, 120151. doi: 10.1016/j.saa.2021.120151
	(b) Zhang, Y. L; Shao, X. M; Wang, Y.; Pan, F. C.; Kang, R. X.; Peng, F. F.; Huang, Z. T.; Zhang, W. J; Zhao, W. L. Chem. Commun. 2015, 51, 4245. doi: 10.1039/C4CC08687B
	(c) Ge, C. P.; Wang, H.; Zhang, B. X.; Yao, J.; Li, X. M; Feng, W. M; Zhou, P. P.; Wang, Y. W; Fang, J. G Chem. Commun. 2015, 51, 14913.
[3]	(a) Xu, Z. Y.; Zhao, X. F.; Zhou, M.; Zhang, Z. J; Qin, T. Y; Wang, D.; Wang, L.; Peng, X. J.; Liu, B. Sens. Actuators, B 2021, 345, 130367. doi: 10.1016/j.snb.2021.130367
	(b) Xu, Z. Y.; Si, S. F.; Zhang, Z. J; Tan, H. Y; Qin, T. Y; Wang, Z. L; Wang, D.; Wang, L.; Liu, B. Anal. Chim. Acta 2021, 1176, 338763. doi: 10.1016/j.aca.2021.338763
[4]	(a) Anbu, S.; Paul, A.; Surendranath, K.; Sidali, A.; Pombeiro, A. J. L. J. Inorg. Biochem. 2021, 220, 111466. doi: 10.1016/j.jinorgbio.2021.111466
	(b) Dimov, S. M.; Georgiev, N. I.; Asiri, A. M.; Bojinov, V. B J. Fluoresc. 2014, 24, 1621.
	(c) Georgiev, N. I.; Dimov, S. M.; Asiri, A. M.; Alamry, K. A.; Obaid, A.Y.; Bojinov, V. B. J. Lumin. 2014, 149, 325. doi: 10.1016/j.jlumin.2014.01.028
[5]	Qu, L. J.; Yin, C. X; Huo, F. J.; Li, J. F; Chao, J. B; Zhang, Y. B. Sens. Actuators, B 2014, 195, 246. doi: 10.1016/j.snb.2014.01.026
[6]	Vasilev, A. A.; Baluschev, S.; Cheshmedzhieva, D.; Ilieva, S.; Castano, O. D.; Vaquero, J. J.; Angelova, S. E.; Landfester, K. Aus. J. Chem. 2015, 68, 1399. doi: 10.1071/CH15139
[7]	Girouard, S.; Houle, M. H.; Grandbois, A.; Keillor, J. W.; Michnick, S. W. J. Am. Chem. Soc. 2005, 127, 559. doi: 10.1021/ja045742x
[8]	Khosravi, A.; Moradian, S.; Gharanjig, K.; Afshar, F. T. J. Chin. Chem. Soc. 2005, 52, 495. doi: 10.1002/jccs.v52.3
[9]	Alexiou, M. S.; Tyman, J. H. P. J. Chem. Res. Miniprint 2000, 5, 632.
[10]	Costi, M. P.; Tondi, D.; Rinaldi, M.; Barlocco, D.; Cignarella, G.; Santi, D. V.; Musiu, C.; Pudu, I.; Vacca, G.; Colla, P. L. Eur. J. Med. Chem. 1996, 31, 1011. doi: 10.1016/S0223-5234(97)86180-6
[11]	Cui, L.; Peng, Z. X.; Ji, C. F.; Huang, J. H. Huang, D. T.; Ma, J.; Zhang, S. P.; Qian, X. H.; Xu, Y. F. Chem. Commun. 2014, 50, 1485. doi: 10.1039/C3CC48304E
[12]	Zhou, J; Fang, C. L; Liu, Y; Zhao, Y; Zhang, N; Liu, X. J.; Wang, F. Y.; Shangguan, D. H. Org. Biomol. Chem. 2015, 13, 3931. doi: 10.1039/C5OB00302D
[13]	Wang, J. F; Jin, S.; Akay, S.; Wang, B. H. Eur. J. Med. Chem., 2007, 13, 2091.
[14]	Yuan, D. W; Brown, R. G.; Hepworth, J. D.; Alexiou, M. S.; Tyman, J. H. P. J. Heterocycl. Chem. 2008, 45, 397. doi: 10.1002/jhet.v45:2
[15]	Li, X. M; Zheng, Y. J; Tong, H. J; Qian, R.; Zhou, L.; Liu, G. X; Tang, Y.; Li, H.; Lou, K. Y.; Wang, W. Chem.-Eur. J. 2016, 27, 9247.