CN114853779B - 细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针、制备方法及应用 - Google Patents
细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针,该探针是可以高选择性和高灵敏度检测生物硫醇的免洗荧光探针,同时还可以对巯基蛋白质进行快速标记。本发明的探针是具有如式I结构所示的3'‑(二乙氨基)‑6'‑羟基‑3‑环内酯‑氧杂蒽环衍生物或3',6'‑2羟基‑3‑环内酯‑氧杂蒽环衍生物:其中R为OH或N(CH3)2。
Description
技术领域
本发明涉及一种探针、制备方法及应用,更具体地说涉及一种细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针、制备方法及应用。
背景技术
生物硫醇包括半胱氨酸(Cys)、同型半胱氨酸(Hcy)、谷胱甘肽(GSH)和含巯基的蛋白质,在细胞功能中蛋白质的合成和折叠、信号转导、解毒、代谢等多种生物和生理过程中发挥着重要作用。此外,硫醇氧化已被报道为许多疾病的潜在病因,如阿尔茨海默病、帕金森病和糖尿病。因此,对生物硫醇,特别是含巯基蛋白的检测,需要准确、灵敏的方法,具有重要意义。然而其他的传统方法通常成本昂贵,样品量大,不能监测细胞内硫醇和快速标记巯基蛋白质。而荧光探针具有灵敏度高、选择性好、实时监测等优点。近年来,特异性检测硫醇荧光探针的策略被大量报道。然而能够同时对生物体内硫醇成像和标记巯基蛋白质的荧光探针却很少,其中许多探针水溶性差、灵敏度低、响应时间长、特异性差,荧光背景高并且在生物应用中存在着较高的生物毒性,这些缺陷很大程度地影响了探针的应用。因此,开发能够快速检测硫醇且免洗的新型荧光探针是非常有必要的。
发明内容
本发明所要解决的技术问题是:克服现有技术存在的不足,提供一种细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针,该探针是可以高选择性和高灵敏度检测生物硫醇的免洗荧光探针,同时还可以对巯基蛋白质进行快速标记。
此外,本发明还提供该细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针的制备方法以及该探针在生物硫醇检测及巯基蛋白质标记中的实际应用。
本发明的技术构思和原理如下:经研究发现3'-羟基-3-环内酯-氧杂蒽环衍生物{3'-(二乙氨基)-6'-羟基-3-环内酯-氧杂蒽环衍生物或3',6'-2羟基-3-环内酯-氧杂蒽环衍生物}探针可选择性与生物硫醇及巯基蛋白质反应并发生颜色和荧光的变化,且在0-10μM生物硫醇的浓度或者0-0.3mg/mL巯基蛋白质(卵清白蛋白OVA)的浓度范围内,557nm处的荧光强度与生物硫醇的浓度有着较好的线性关系。本发明首次制备3'-羟基-3-环内酯-氧杂蒽环衍生物并首次将其用于生物硫醇的选择性检测,并对细胞内及生物体内的生物硫醇进行快速检测成像,并且可以快速标记巯基蛋白质,从而解决了现有技术存在的问题。
本发明的技术方案如下:
本发明的细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针,是具有如式I结构所示的3'-(二乙氨基)-6'-羟基-3-环内酯-氧杂蒽环衍生物或3',6'-2羟基-3-环内酯-氧杂蒽环衍生物:
其中R为OH或N(CH3)2。
本发明上述的细胞及生物体内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针的制备方法,包括以下步骤:
将3'-羟基-3-环内酯-氧杂蒽环-4-甲醛、2-环戊烯-1-酮和咪唑混合于四氢呋喃及去离子水中,反应生成的3',6'-2羟基-3-环内酯-氧杂蒽环衍生物即为细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针;
或者,将3'-(二乙氨基)-6'-羟基-3-环内酯-氧杂蒽环-4-甲醛、2-环戊烯-1-酮和咪唑混合于四氢呋喃及去离子水中,反应生成的3'-(二乙氨基)-6'-羟基-3-环内酯-氧杂蒽环衍生物即为细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针。
本发明上述的细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针的制备方法,其进一步的技术方案是所述反应条件为室温条件下,搅拌反应72h以上;反应结束后进行纯化,再真空干燥即得细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针。再进一步的技术方案是所述的反应结束后进行纯化具体步骤为:将反应液减压浓缩,粗产物在硅胶柱上用二氯甲烷/甲醇进一步纯化。
本发明上述的荧光探针在用于细胞内生物硫醇的检测与成像以及特异性快速标记巯基蛋白质中的应用。
本发明具有以下有益效果:
1)本发明荧光探针在pH=7.4的HEPES缓冲溶液中几乎没有荧光,与生物硫醇发生1,4-Michael加成反应后,由于电子转移,螺环打开,增大共轭结构,吸收发生变化,荧光显著增强。
2)采用本发明荧光探针后,检测灵敏度高,对生物硫醇的检测限可达到10-7M。且反应迅速,表观反应速率常数可达到10min-1。
3)采用本发明荧光探针后,检测灵敏度高,可快速标记巯基蛋白质,对巯基蛋白质的检测限可达到10-3mg/mL。与商品化的荧光染料(荧光素-5-马来酰亚胺)相比,在相同测试条件下,本探针对含巯基的蛋白质荧光响应特异性更强,信噪比更高,稳定性更好。本探针具有作为特异性标记巯基蛋白质的荧光染料的优异性能,可广泛应用于巯基蛋白质的标记与检测。
4)本发明荧光探针仅与生物硫醇发生荧光反应,对其它氨基酸均无反应,具有很好的选择性和特异性。具有适宜的荧光发射波长(557nm及520nm)。
5)本发明荧光探针自身在pH为2-10之间无荧光,生物硫醇的加入引起的荧光增强在pH为7-9之间达到最大值。适合生物系统的应用。
6)本发明荧光探针对生物体具有低毒性,且对细胞内或斑马体内生物硫醇进行荧光成像。
附图说明
图1为实施例3中本发明的荧光探针与不同氨基酸反应的荧光光谱。
图2为实施例3中本发明的荧光探针与不同氨基酸反应的吸收光谱图。
图3-5为实施例4中本发明的荧光探针分别与Cys、GSH、Hcy的荧光增量图。
图6-8为实施例4中本发明的荧光探针对分别与Cys、GSH、Hcy的吸收增量图。
图9-11为实施例4中本发明的荧光探针对不同pH溶液中,分别加入Cys、GSH、Hcy后,荧光强度增强与pH值的关系图。
图12-14为实施例5中本发明的荧光探针与Cys、GSH、Hcy反应后,荧光强度随时间的变化图。
图15为实施例6中本发明的荧光探针对细胞毒性实验图。
图16为实施例7中本发明的荧光探针对细胞内生物硫醇成像的细胞荧光图。
图17为实施例8中本发明的荧光探针对巯基蛋白质特异性识别的荧光光谱图。
图18为实施例8中本发明的荧光探针与卵清白蛋白OVA的荧光增量图。
图19为实施例8中本发明的荧光探针与蛋白质反应后凝胶电泳图。
图20-21为实施例9中商品化巯基荧光染料(荧光素-5-马来酰亚胺)和本发明的荧光探针,分别与各种蛋白质反应后的荧光增量对比图以及2小时后的荧光光谱变化图。
图22为实施例9中商品化巯基荧光染料(荧光素-5-马来酰亚胺)与蛋白质反应后的凝胶电泳图。
图23-24为实施例10中本发明的荧光探针在细胞及生物体内硫醇成像得荧光图。
图25为实施例1中本发明的荧光探针1H-NMR图
图26为实施例1中本发明的荧光探针高分辨质谱图
图27为实施例2中本发明的荧光探针1H-NMR图
图28为实施例2中本发明的荧光探针高分辨质谱图
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。
实施例1制备细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针
将415mg 3'-(二乙氨基)-6'-羟基-3-环内酯-氧杂蒽环-4-甲醛、164mg 2-环戊烯-1-酮和18mg咪唑混合于15mL四氢呋喃和10mL去离子水中,在室温条件下,搅拌反应72h。反应结束后,将反应液减压浓缩,粗产物在硅胶柱上用二氯甲烷/甲醇进一步纯化,真空干燥即得淡红色固体148mg,即为细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针(1H-NMR图和高分辨质谱图见图25、图26)。所得荧光探针纯品实测分子量为479.17。
本实施例工艺路线:
实施例2制备细胞内生物硫醇检测与特异性快速标记巯基蛋白质的荧光探针
将360mg 3'-羟基-3-环内酯-氧杂蒽环-4-甲醛、164mg 2-环戊烯-1-酮和18mg咪唑混合于15mL四氢呋喃及10mL去离子水中,在室温条件下,搅拌反应72h。反应结束后,将反应液减压浓缩,粗产物在硅胶柱上用乙酸乙酯/石油醚进一步纯化,真空干燥即得淡黄色固体,即为为细胞内生物硫醇检测与体内硫醇成像的荧光探针。(1H-NMR图和高分辨质谱图见图27、图28)。所得荧光探针纯品实测分子量为423.0869。
本实施例工艺路线:
实施例3本发明的荧光探针与各种氨基酸反应的光谱性质
称取4.79mg实施例1制得生物硫醇和巯基蛋白质标记的荧光探针,配成浓度为1mM的10mL CH3CN溶液,作为母液。
荧光光谱及吸收光谱测试:将30μL上述母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中,然后分别加入待测物:Cys、Hcy、GSH、Arg、Gly、Ile、Leu、Pro、Val、Phe、Trp、Tyr、Asp、Glu、Arg、His、Lys、Ser、Thr、Asn、Gln、Met,使待测物终浓度为100μM,荧光探针终浓度为10μM。在520nm激发光波长下即时测试其荧光发射光谱。激发与发射的狭缝宽度为3/1.5nm。所得荧光光谱图及吸收光谱分别如图1和图2所示。
以上结果表明:
(1)实施例1制得荧光探针本身在溶液中几乎没有荧光,但随生物硫醇的加入,该探针在557nm处荧光发射增强约80倍。
(2)实施例1制得荧光探针本身在溶液中呈橘黄色,在494和525nm处有两个主要吸收带,随着生物硫醇的加入,494nm处的吸收带逐渐消失,525nm处的吸收带明显增强。溶液颜色由橘黄色变为粉红色。
(3)实施例1制得荧光探针对生物硫醇具有高度的选择性和特异性,并且在上述条件下,能够从Arg、Gly、Ile、Leu、Pro、Val、Phe、Trp、Tyr、Asp、Glu、Arg、His、Lys、Ser、Thr、Asn、Gln、Met等氨基酸中区分出生物硫醇(Cys、Hcy、GSH)。
实施例4本发明的荧光探针与生物硫醇反应产物的光谱性质
将30μL实施例2中的母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中,然后分别加入不同当量的Cys、Hcy、GSH,使荧光探针的终浓度为10μM,生物硫醇浓度分别为0μM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM。生物硫醇加入后,即时测量其荧光发射光谱。荧光发射光谱测定时以520nm激发波长;激发与发射的狭缝宽度为3/1.5nm。所得荧光强度光谱增量图见图3-5;所得吸收光谱图见图6-8。
将30μL实施例1中的母液加入到一定量的不同pH溶液中,pH值分别为2,3,4,5,6,7,8,9,10。然后分别加入Cys、Hcy、GSH,使荧光探针的终浓度为10μM,生物硫醇浓度分别为100μM。生物硫醇加入后,即时测量其荧光发射光谱。荧光发射光谱测定时以520nm激发波长;激发与发射的狭缝宽度为3/1.5nm。所得荧光强度-pH图见图9-11。
该实验结果表明,反应后荧光强度随生物硫醇浓度的增加而增加;反应后荧光强度与0-10μM范围内的生物硫醇浓度呈较好的线性关系,可以用于生物硫醇含量的定量分析检测。荧光探针自身在pH为2-10之间无荧光,生物硫醇的加入引起的荧光增强在pH为7-9之间达到最大值。适合生物系统的应用。
实施例5本发明的荧光探针与生物硫醇反应速率计算
将30μL实施例1中的母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中,然后分别加入不同当量的Cys、Hcy、GSH,使荧光探针的终浓度为10μM,生物硫醇浓度分别为100μM。生物硫醇加入后,即时测量其荧光动力学曲线。动力学曲线测定时以520nm激发波长,557nm发射波长;激发与发射的狭缝宽度为3/1.5nm。所得动力学曲线见图12-14。
该实验结果表明,探针与生物硫醇在15s内反应完全,且计算得表观速率常数约为10min-1。
实施例6本发明的荧光探针对细胞毒性的测定
将实施例1中的母液分别稀释到0μM,60μM,150μM,300μM后,分别添加10μM到接种了已贴壁的HeLa细胞的96孔板中,此时探针的浓度分别为0μM,2μM,5μM和10μM。在孵化12小时后,分别添加20μL甲基噻唑四唑(MTT)(0.5mg/mL),在37℃下,5%CO2条件下培养4小时,然后移除多余的MTT溶液,加入DMSO(100μL)。采集490nm处的吸收,并计算得到各浓度下的细胞存活率。实验结果如图15所示,该实验结果表明,探针对活细胞具有低毒性。
实施例7本发明的荧光探针对细胞内硫醇的成像
称取4.79mg实施例1制得生物硫醇和巯基蛋白质标记的荧光探针,配成浓度为2.5mM的4mL CH3CN溶液,作为母液。
取探针母液2μL加入到1mL的培养基中,此时探针的浓度为5μM,37℃,5%CO2条件下培养HeLa细胞10min;分别取Cys(10mM),GSH(10mM)及巯基掩蔽剂N-ethylmaleimide(NEM)(10mM)各10μL加入到1mL的培养基中,此时外源性生物硫醇的浓度和巯基掩蔽剂的浓度为100μM。用倒置荧光显微镜对HeLa细胞进行细胞成像,实验结果如图16所示。图(A)表示荧光探针对HeLa细胞内源性生物硫醇的荧光成像图;图(B)表示在外源性Cys加入后HeLa细胞中的荧光成像图;图(C)表示在外源性GSH加入后HeLa细胞中的荧光成像图;图(D)表示加入巯基掩蔽剂NEM后HeLa细胞中的荧光成像图。
除上述实施例外,本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案,均落在本发明要求的保护范围。
实施例8本发明的荧光探针对巯基蛋白质的标记
称取蛋白质(牛血清白蛋白BSA、卵清白蛋白OVA及作为对比的木聚糖酶Xyn)各10mg溶于1mL的去离子水中,配成10mg/mL的蛋白质溶液。
荧光光谱测试:将30μL实施例1中的母液加入到一定量的10mM HEPES缓冲溶液(pH7.4)中,然后分别加入蛋白质:BSA,OVA和Xyn,使待测物终浓度为0.3mg/mL,荧光探针终浓度为10μM。在520nm激发光波长下即时测试其荧光发射光谱。激发与发射的狭缝宽度为3/1.5nm。所得荧光光谱图如图17所示。
将30μL实施例1中的母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中,然后分别加入不同当量的OVA,使荧光探针的终浓度为10μM,OVA浓度分别为0mg/mL,0.03mg/mL,0.06mg/mL,0.09mg/mL,0.12mg/mL,0.15mg/mL,0.18mg/mL,0.21mg/mL,0.24mg/mL,0.27mg/mL,0.3mg/mL。OVA加入后,即时测量其荧光发射光谱。荧光发射光谱测定时以520nm激发波长;激发与发射的狭缝宽度为3/1.5nm。所得荧光强度光谱增量图见图18。
将各蛋白质稀释到0.1mg/mL,分别与实施例1中的母液以1:1的比例混合形成探针与蛋白质加合物,用12% SDS-PAGE进行分离和分析。加合物和上样缓冲液的混合物煮沸5分钟后,每孔上样15μL,电泳电压120V。凝胶荧光成像在Tanon-5200Multi凝胶成像仪中,绿光激发下拍摄。作为对照,采用考马斯亮蓝染色后,在302nm透射光下成像。所得蛋白质凝胶电泳分析图见图19。
实施例9对比商品化的巯基染料对小分子的响应与对含巯基蛋白质的标记。
称取4.28mg商品化的巯基染料,配成浓度为1mM的10mL DMSO溶液,作为母液。
荧光光谱测试:将30μL上述巯基染料的母液或实施例7中的母液加入到一定量的10mM HEPES缓冲溶液(pH 7.4)中,然后分别加入蛋白质:BSA,OVA和Xyn,使待测物终浓度为0.3mg/mL,荧光探针终浓度为10μM。在520nm和480nm的激发波长下分别即时测试其荧光发射光谱。且收集实上述巯基染料与实施例7中的母液在HEPES缓冲溶液中,在520nm和480nm的激发波长下,557nm和520nm处的荧光强度-时间的光谱图。激发与发射的狭缝宽度为3/1.5nm。所得荧光强度增量光谱图如图20-21所示。
将各蛋白质稀释到0.1mg/mL,分别与上述巯基染料的母液以1:1的比例混合形成探针与蛋白质加合物,用12%SDS-PAGE进行分离和分析。加合物和上样缓冲液的混合物煮沸5分钟后,每孔上样15μL,电泳电压120V。凝胶荧光成像在Tanon-5200Multi凝胶成像仪中,蓝光激发下拍摄。作为对照,采用考马斯亮蓝染色后,在302nm透射光下成像。所得蛋白质凝胶电泳分析图见图22。
经分析以上结果表明:
(1)实施例1制得荧光探针对巯基蛋白质具有高度的选择性和特异性,并且在上述条件下,能够与巯基蛋白质反应后荧光增强90倍左右。
(2)实施例1制得荧光探针随OVA浓度的增加而增加;反应后荧光强度与0-0.3mg/mL范围内的OVA浓度呈较好的线性关系,可以用于巯基蛋白质OVA含量的定量分析检测,并计算得到对OVA的检测限低至1.51μg/mL。
(3)实施例1制得荧光探针是一种免洗探针,可以对巯基蛋白质进行快速特异性标记。
(4)实施例1制得荧光探针相较于商品化的巯基染料对蛋白质的标记更具特异性,且稳定性更好。
实施例10本发明探针在生物体内对硫醇的成像。
称取4.23mg实施例2制得生物硫醇荧光探针,配成浓度为2mM的5mL DMSO溶液,作为母液。
取探针母液10μL加入到1mL的培养基中,此时探针的浓度为20μM,与小鼠P19胚胎癌细胞和斑马鱼共孵育30分钟;取巯基掩蔽剂N-ethylmaleimide(NEM)(10mM)各40μL加入到1mL的培养基中,此时巯基掩蔽剂的浓度为100μM,孵育20分钟后加入探针再孵育30分钟。用共聚焦显微镜对小鼠P19胚胎癌细胞进行细胞成像。观察与探针(5μM)共孵育1小时的斑马鱼,并进行生物体内成像,实验结果如图23-24所示。
除上述实施例外,本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案,均落在本发明要求的保护范围。
Claims (1)
1.一种具有如式I结构所示的化合物在制备用于细胞或生物体内生物硫醇的检测与成像以及特异性快速标记巯基蛋白质的荧光探针中的应用;
其中R为N(CH3)2。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5352803A (en) * | 1992-03-30 | 1994-10-04 | Abbott Laboratories | 5(6)-methyl substituted fluorescein derivatives |
KR20110090417A (ko) * | 2010-02-03 | 2011-08-10 | 이화여자대학교 산학협력단 | 티올 선택성을 갖는 플루오레세인 유도체를 포함하는 형광 센서 및 이를 이용한 티올 검출방법 |
CN102964353A (zh) * | 2012-10-25 | 2013-03-13 | 南京工业大学 | 用于检测乙酰胆碱酯酶活性抑制率的探针、用途及制备方法 |
CN103289681A (zh) * | 2013-06-09 | 2013-09-11 | 南京工业大学 | 谷胱甘肽荧光探针及其制备方法和用途 |
KR20150090673A (ko) * | 2014-01-29 | 2015-08-06 | 한국과학기술원 | 플루오레세인을 기반으로 한 시스테인/호모시스테인에 선택적으로 반응하는 화합물 및 형광 프로브 |
CN114149448A (zh) * | 2021-11-12 | 2022-03-08 | 山东第一医科大学(山东省医学科学院) | 一种用于检测硝基还原酶的近红外荧光探针及应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8569516B2 (en) * | 2001-09-07 | 2013-10-29 | Elitech Holding B.V. | Compounds and methods for fluorescent labeling |
US6972339B2 (en) * | 2001-09-07 | 2005-12-06 | Epoch Biosciences, Inc. | Compounds and methods for fluorescent labeling |
WO2004094647A2 (en) * | 2003-04-18 | 2004-11-04 | Cytovia, Inc. | Methods of treating diseases responsive to induction of apoptosis and screening assays |
US7705040B2 (en) * | 2005-10-07 | 2010-04-27 | The University Of Hong Kong | Reagents for highly specific detection of peroxynitrite |
-
2022
- 2022-06-09 CN CN202210648870.4A patent/CN114853779B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5352803A (en) * | 1992-03-30 | 1994-10-04 | Abbott Laboratories | 5(6)-methyl substituted fluorescein derivatives |
KR20110090417A (ko) * | 2010-02-03 | 2011-08-10 | 이화여자대학교 산학협력단 | 티올 선택성을 갖는 플루오레세인 유도체를 포함하는 형광 센서 및 이를 이용한 티올 검출방법 |
CN102964353A (zh) * | 2012-10-25 | 2013-03-13 | 南京工业大学 | 用于检测乙酰胆碱酯酶活性抑制率的探针、用途及制备方法 |
CN103289681A (zh) * | 2013-06-09 | 2013-09-11 | 南京工业大学 | 谷胱甘肽荧光探针及其制备方法和用途 |
KR20150090673A (ko) * | 2014-01-29 | 2015-08-06 | 한국과학기술원 | 플루오레세인을 기반으로 한 시스테인/호모시스테인에 선택적으로 반응하는 화합물 및 형광 프로브 |
CN114149448A (zh) * | 2021-11-12 | 2022-03-08 | 山东第一医科大学(山东省医学科学院) | 一种用于检测硝基还原酶的近红外荧光探针及应用 |
Non-Patent Citations (3)
Title |
---|
A thiol-specific fluorescent probe and its application for bioimaging;Xiaoqiang Chen et al.;《Chem. Commun.》;第46卷;第2751页 * |
硫醇类荧光探针研究进展;尹伶灵等;《分析化学》;第37卷(第07期);第1073-1081页 * |
选择性生物小分子硫醇荧光探针的研究进展;谢光杰等;《化学研究与应用》;第28卷(第09期);第1177-1185页 * |
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