朱東建,江 華,2*
(1.北京分子科學(xué)國(guó)家實(shí)驗(yàn)室,中國(guó)科學(xué)院 化學(xué)研究所 光化學(xué)重點(diǎn)實(shí)驗(yàn)室,北京100190;2.北京師范大學(xué) 化學(xué)學(xué)院,北京100875)
生物體內(nèi)的硫醇如半胱氨酸(Cys)、同型半胱氨酸(Hcy)、谷胱甘肽(GSH)在生理和病理過程中起至關(guān)重要的作用[1-2]。細(xì)胞內(nèi)硫醇水平的改變與很多疾病密切相關(guān)。體內(nèi)缺乏半胱氨酸(Cys)會(huì)導(dǎo)致多種病癥,如兒童生長(zhǎng)緩慢,肝損傷和皮膚損傷等[3-5]。血液中同型半胱氨酸(Hcy)的濃度增加會(huì)導(dǎo)致維生素B12的缺失和老年癡呆癥[6-7]。谷 胱 甘 肽 (GSH)在 細(xì) 胞 內(nèi) 含 量 在 1 mmol/L到15mmol/L 之間[8],是細(xì)胞內(nèi)最富裕的硫醇,在維持細(xì)胞的氧化還原動(dòng)態(tài)平衡中起著重要作用[9]。因此,檢測(cè)生物體系中硫醇含量具有非常重要的意義。
目前,用于檢測(cè)硫醇的方法有很多,如高效液相色譜法[10-11]、電化學(xué)法[12-13]、熒光法等。相比其他方法,熒光法由于具有選擇性好、靈敏度高、快速簡(jiǎn)便等優(yōu)點(diǎn),因此,近年發(fā)展了許多檢測(cè)硫醇的熒光探針[14-20]。由于生物樣品基體的自身熒光波長(zhǎng)一般小于600nm[21],而大多數(shù)探針的熒光發(fā)射波長(zhǎng)與生物樣品的背景熒光有重疊,因此極大地限制了這類熒光分析法靈敏度,而在近紅外熒光(λem>600nm)光區(qū),生物樣品基體光吸收或熒光強(qiáng)度很小,因而背景干擾大大降低。因此,發(fā)展近紅外硫醇探針是一項(xiàng)具有挑戰(zhàn)性的工作。目前有少量近紅外硫醇探針通過單個(gè)熒光信號(hào)強(qiáng)度的增加來達(dá)到檢測(cè)目的[22-23],而熒光強(qiáng)度很容易受到其他因素(如樣品環(huán)境條件、探針濃度等)的影響,因此,此類探針并不能提供足夠的精度以進(jìn)行定量的檢測(cè)。具有自校正作用的、通過兩個(gè)波長(zhǎng)的變化指示識(shí)別過程的比率測(cè)量型熒光探針則能很好的實(shí)現(xiàn)定量檢測(cè)[24]。
本工作根據(jù)我們以前設(shè)計(jì)的近紅外比率熒光探針的原理[25],設(shè)計(jì)合成了以七甲川花菁為熒光發(fā)色團(tuán)、二硫鍵為硫醇的特異性識(shí)別位點(diǎn)的硫醇近紅外比率熒光探針分子CySS。
UV-2500紫外-可見分光光度計(jì)(日本島津公司),F(xiàn)-4600熒光分光光度計(jì)(日本日立公司),Bruker AVANCE-400核磁共振儀(美國(guó) Bruker公司),APEXII型FT-ICR質(zhì)譜儀(美國(guó)Bruker公司),F(xiàn)V-1000共聚焦顯微鏡(日本 Olympus)。
細(xì)胞實(shí)驗(yàn)中所用NIH 3T3細(xì)胞(鼠成纖維細(xì)胞),標(biāo)準(zhǔn)PBS緩沖液購(gòu)于協(xié)和醫(yī)科大學(xué)細(xì)胞中心;培養(yǎng)基DMEM(Hyclone,American),標(biāo)準(zhǔn)胎牛血清FBS(Hyclone,American),雙抗(青霉素,鏈霉素;Hyclone,American)購(gòu)于北京拜爾迪生物技術(shù)有限公司。培養(yǎng)基的配置:DMEM溶液,10%的FBS,1%的雙抗,搖勻,4℃保存。
所有合成原料與溶劑均為市售分析純。半胱氨酸,同型半胱氨酸,谷胱甘肽,二硫蘇糖醇(DTT),2-氨基乙硫醇(2-AET),2-巰基乙酸(2-MEA),葡 萄 糖 (Glu),丙 氨 酸 (Ala),纈 氨 酸(Val),絲氨酸(Ser),組氨酸(His),亮氨酸(Leu),賴氨酸(Lys),2-氨基乙酸(2-AEA),KCl,NaCl,CaCl2,MgSO4溶解在超純水中制得溶液。柱色譜硅膠為煙臺(tái)化學(xué)工業(yè)研究所產(chǎn)品(200~300目)。
探針CySS溶于DMSO配制成濃度為2.0 mmol/L的溶液,并于4℃下保存;紫外可見及熒光光譜均在緩沖溶液(100mmol/L HEPES,100 mmol/L NaCl,pH =7.4,DMSO∶水=5∶95(體積比))中測(cè)定;熒光量子產(chǎn)率的測(cè)定以Cardiogreen在 DMSO(Φ=0.13)作為熒光標(biāo)準(zhǔn)物[26]。
NIH 3T3細(xì)胞以配置好的DMEM 培養(yǎng)基(10%FBS,1% 雙抗),在25cm3的細(xì)胞培養(yǎng)瓶中,置于37℃培養(yǎng)箱中培養(yǎng),每?jī)商鞊Q一次新鮮的培養(yǎng)基;將細(xì)胞培養(yǎng)在35mm適合倒置共聚焦熒光顯微鏡測(cè)試的表面皿中,孵化48h,細(xì)胞貼壁狀態(tài)良好后,在做成像實(shí)驗(yàn)前更換為無血清、無雙抗的DMEM培養(yǎng)基進(jìn)行染色。
在共聚焦顯微鏡下進(jìn)行細(xì)胞熒光成像實(shí)驗(yàn)前,吸出培養(yǎng)基并用PBS沖洗三次,再加入1mL PBS。激發(fā)波長(zhǎng)為635nm,熒光收集窗口700~770nm和780~800nm。
CySS的合成路線如圖1所示。
圖1 CySS的合成路線和可能的反應(yīng)機(jī)理The synthetic route of CySS and the possible reaction mechanism for thiols
在三口瓶中加入1.59g(15mmol)無水碳酸鈉,高溫除水后冷卻到室溫,抽換氣三次后,在氬氣保護(hù)下加入5mL無水甲苯,冰浴至0℃左右,滴加888.72mg(3mmol)三光氣的甲苯溶液,滴加完攪拌15min,滴加294mg(1.5mmol)1[27]的甲苯溶液,滴加完緩慢升至室溫反應(yīng)3h后,用氬氣趕走剩余的光氣后,過濾抽干后,將其用10 mL無水二氯甲烷溶解,低溫緩慢滴加到170mg(0.25mmol)CyN[25]和0.75mL (4.5mmol)N,N′-二異丙基乙胺(DIEA)的二氯甲烷溶液中,加完緩慢升至室溫反應(yīng)過夜,TLC檢測(cè)有產(chǎn)物生成。反應(yīng)完畢后用1mol/L鹽酸洗兩次,飽和氯化鈉液洗一次,有機(jī)相用無水硫酸鈉干燥,過濾旋干后,用硅膠色譜柱提純得到綠色目標(biāo)化合物CySS 45mg。產(chǎn)率20%。1HNMR (400MHz,CD2Cl2):7.42~7.37(m,6H),7.35~7.31(t,J=7.2Hz,2H),7.29~7.27(d,J=7.6Hz,2H),7.25~7.22(t,J=7.2Hz,3H),7.19~7.17(d,J=8.0 Hz,2H),6.10~6.06(d,J=14.0Hz,2H),4.76(s,2H),4.27~4.24(t,J=6.4Hz,2H),4.19~4.16(t,J=6.4Hz,2H),3.60(s,6H),2.81~2.76(m,5H),2.59~2.53(m,3H),2.07~2.04(m,2H),1.96(s,3H),1.26(s,12H)。13CNMR(400MHz,CD2Cl2):172.63,170.69,155.84,154.67,143.21,142.43,141.13,136.97,130.81,129.14,128.94,128.69,128.31,125.36,122.30,111.05,101.54,64.24,62.35,55.75,49.25,37.83,37.59,32.08,28.18,27.72,25.35,21.12,20.85。HRMS(ESI)for C46H54N3O4S2+([M-I]+):calcd:776.35503,found:776.35385。
圖2 CySS加入半胱氨酸的紫外(a)和熒光(b)光譜(a)Absorption spectra of CySS(1μmol/L)0,15,30,45,60,90and 120min after addition of Cys(2mmol/L).(b)Fluorescence spectra of CySS(2μmol/L)every 5min within 2hafter addition of Cys(3mmol/L),λex= 676nm
如圖 2(a)所示,1μmol/L CySS在 HEPES緩沖液中的最大吸收峰大約在785nm(ε=1.81×105L·mol-1·cm-1),向該緩沖溶液中加入2 mmol/L的半胱氨酸后,我們可以觀察到,隨著時(shí)間的變化785nm處的吸收強(qiáng)度減弱,與之同時(shí)在645nm處一個(gè)新的吸收峰不斷增強(qiáng),與CyN在HEPES緩沖液中的最大吸收峰一樣[25],而且兩者之間的吸收波長(zhǎng)相差達(dá)到140nm。這些數(shù)據(jù)表明,當(dāng)半胱氨酸加入到該反應(yīng)體系后,探針分子CySS上面的二硫鍵與半胱氨酸反應(yīng),并且經(jīng)過分子內(nèi)進(jìn)攻反應(yīng)使酰胺鍵斷裂釋放出產(chǎn)物分子CyN,與我們提出的反應(yīng)機(jī)理一致(如圖1所示)。同時(shí),我們也研究了熒光發(fā)射光譜的變化情況,如圖2(b)所示,用676nm 激發(fā),2mol/L CySS在HEPES緩沖液中的最大熒光發(fā)射峰大約在808 nm (Φ=0.035),向該緩沖溶液中加入3mmol/L的半光氨酸后,我們可以觀察到,隨著時(shí)間的變化808nm處的熒光發(fā)射強(qiáng)度逐漸減弱,與之同時(shí)在747nm處一個(gè)新的熒光發(fā)射峰不斷增強(qiáng),與CyN在HEPES緩沖液中的最大熒光發(fā)射峰相同[25],而且兩者之間的吸收波長(zhǎng)相差達(dá)到61nm,并且在793nm處產(chǎn)生一個(gè)等發(fā)射點(diǎn)。熒光發(fā)射光譜的變化同樣證明了上述反應(yīng)機(jī)理。
CySS對(duì)硫醇有選擇性識(shí)別的效果。我們向2 μmol/L CySS的HEPES緩沖溶液中分別加入3 mmol/L各種巰基化合物,包括半胱氨酸、同型半胱氨酸、谷胱甘肽、二硫蘇糖醇、2-氨基乙硫醇、2-巰基乙酸,2h后對(duì)其熒光光譜進(jìn)行測(cè)試,結(jié)果表明,747nm和808nm處的熒光發(fā)射強(qiáng)度之間的比率(F747nm/F808nm)發(fā)生了明顯變化,反應(yīng)前后比率(F747nm/F808nm)從0.06變化到2.9~4.3,分別由圖3(a)灰黑條表示。而其它不含巰基的氨基酸(丙氨酸、纈氨酸、絲氨酸、組氨酸、亮氨酸、賴氨酸)、葡萄糖、2-氨基乙酸和常見的金屬離子(K+、Na+、Ca2+、Mg2+)3mmol/L,反應(yīng)2h都沒有引起明顯的熒光發(fā)射強(qiáng)度比率(F747nm/F808nm)上的改變,如圖3(b)灰條所示。然后再當(dāng)向其中加入3mmol/L半胱氨酸反應(yīng)2h,熒光發(fā)射強(qiáng)度比率(F747nm/F808nm)仍可以看到明顯變化,并且與直接加入3mmol/L半胱氨酸的比率值接近,如圖3(b)黑條所示,說明上述化合物對(duì)檢測(cè)硫醇沒有影響。上述結(jié)果說明,探針分子CySS在近紅外區(qū)域?qū)α虼季哂袠O好的化學(xué)選擇性。
圖3 CySS的化學(xué)選擇性(a)Fluorescence responses of CySS toward various thiols.The gray and black bars represent emission intensity ratios F747nm/F808nmof CySS(2μmol/L)before and 120min after addition of the thiols(3mmol/L),respectively.(b)Fluorescence responses of CySS toward other analytes.The gray and black bars represent emission intensity ratios F747nm/F808nm of CySS(2μmol/L)120min after addition of various analytes(3mmol/L)and further 120min after addition of Cys(3mmol/L),respectively.λex=676nm
在生理pH范圍6~8內(nèi),CySS在808nm處的熒光發(fā)射強(qiáng)度和CyN在747nm處的熒光發(fā)射強(qiáng)度幾乎不受pH的影響,如圖4(a)所示;它們的比率(F747nm/F808nm)也同樣都不受pH的影響,如圖4(b)所示。也就是說,在生理范圍內(nèi),CySS對(duì)硫醇的比率測(cè)量型檢測(cè)并不受到酸堿環(huán)境改變的影響。
對(duì)CySS在水溶液中的應(yīng)用做了上述研究之后,我們接下來將它應(yīng)用到活細(xì)胞成像中。如圖5所示,將 NIH 3T3細(xì)胞用10μmol/L CySS在37℃下培養(yǎng)30min后,我們發(fā)現(xiàn),CySS具有良好的細(xì)胞膜穿透性,已經(jīng)滲透到細(xì)胞內(nèi)部,并且700~770nm熒光發(fā)射窗口和長(zhǎng)波窗口780~800nm處都有熒光(圖5上部);當(dāng)NIH 3T3細(xì)胞用500 μmol/L硫 辛 酸 培 養(yǎng) 一 天 后,再 用 10μmol/L CySS在37℃下培養(yǎng)30min后,進(jìn)行成像實(shí)驗(yàn),在窗口700~770nm處觀察到熒光有明顯的增強(qiáng)(圖5中部);這與以前文獻(xiàn)報(bào)道的硫辛酸會(huì)使細(xì)胞內(nèi)谷胱甘肽濃度增加結(jié)果一致[28];當(dāng)NIH 3T3細(xì)胞用100μmol/L N-乙基馬來酰亞胺(NEM)培養(yǎng)30min后,再用10μmol/L CySS在37℃下培養(yǎng)30min進(jìn)行成像實(shí)驗(yàn),在窗口700~770nm處觀察到熒光明顯減弱(圖5底部),這是因?yàn)镹EM作為硫醇的清除劑[29],使細(xì)胞內(nèi)硫醇濃度降低所致。這說明CySS熒光發(fā)射的變化確實(shí)為細(xì)胞內(nèi)的硫醇濃度變化所引起,而且通過對(duì)兩個(gè)監(jiān)測(cè)窗口做比率圖像,我們可以更加直觀、靈敏地檢測(cè)細(xì)胞內(nèi)硫醇濃度的變化。因此,CySS可以成功應(yīng)用于檢測(cè)細(xì)胞內(nèi)的硫醇濃度的變化,具有很好的應(yīng)用前景。
圖4 pH對(duì)CySS和CyN兩個(gè)波長(zhǎng)熒光強(qiáng)度(a)和比率(b)的影響(a)Effect of pH on fluorescence intensity at 747nm for CyN and 808nm for CySS.(b)Effect of pH on fluorescence intensity ratio of F747nm/F808nmfor CyN and CySS.λex= 676nm
圖5 將CySS用于NIH 3T3細(xì)胞中硫醇成像實(shí)驗(yàn)(a)明場(chǎng)圖像;(b)700~770nm熒光發(fā)射窗口的熒光信號(hào);(c)780~800nm熒光發(fā)射窗口的熒光信號(hào);(d)圖(b)與(c)的比率圖像Confocal fluorescence images of intracellular thiols in NIH 3T3cells with CySS(a)Bright-field transmission images.(b)Fluorescence images with emission collected at 700-770nm.(c)Fluorescence images with emission collected at 780-800nm.(d)Ratiometric images generated from (b)and(c)
本文基于硫醇與二硫鍵的選擇性反應(yīng),設(shè)計(jì)合成了以七甲川花菁為熒光發(fā)色團(tuán)的硫醇近紅外比率熒光探針CySS,并對(duì)其性質(zhì)和應(yīng)用做了詳細(xì)深入的研究。探針分子CySS具有靈敏度高,選擇性好,且不受pH影響等優(yōu)點(diǎn),被成功應(yīng)用到細(xì)胞內(nèi)硫醇的檢測(cè)。
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