CN106749402A - 一种稳定同位素标记手性磷试剂及其制备方法与应用 - Google Patents
一种稳定同位素标记手性磷试剂及其制备方法与应用 Download PDFInfo
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Abstract
一种稳定同位素标记手性磷试剂及其制备方法与应用,涉及手性磷试剂。制备方法:步骤1:稳定同位素标记的醇1的制备;步骤2:稳定同位素标记的亚磷酸酯2的制备;步骤3:稳定同位素标记的N‑磷酰化氨基酸3的制备;步骤4:稳定同位素标记N‑磷酰化氨基酸活化酯4的制备。所述稳定同位素标记手性磷试剂在对含有氨基手性化合物的手性比例进行定量分析;对含有氨基代谢物,如氨基酸、小肽或多肽等进行稳定同位素磷试剂标记定量质谱分析;对生物样品中含有氨基的代谢物进行定量质谱分析中的应用。反应时间短,标记具有高效和高选择性,样品处理步骤简单。
Description
技术领域
本发明涉及手性磷试剂,尤其是涉及一种稳定同位素标记手性磷试剂及其制备方法与应用。
背景技术
稳定同位素化学标记策略与质谱技术联用在小分子代谢物和蛋白生物大分子的定性和定量中发挥着越来越重要的作用,特别是在疾病生物标志物的发现方面。多种基于质谱的稳定同位素化学标记方法及试剂得到广泛开发,如还原甲基化标记、丙酮标记、和丹磺酰氯标记等。[1-3]N-磷酰化氨基酸作为“核酸与蛋白共起源”小分子模型可以发生自组装成肽、成酯、磷上酯交换及磷酰基转位等仿生化反应。近年来,磷酰化标记策略被应用于氨基酸、多肽及蛋白质的定性与定量质谱分析。[4,5]磷酰化标记不但可以提高质谱分析灵敏度,同时有利于色谱分离,特别是针对微量复杂成分的色谱-质谱联用分析。本研究基于N-磷酰化氨基酸独特的物理化学性质,合成了一系列稳定同位素标记N-磷酰化氨基酸活化酯,并基于质谱对含有氨基的小分子代谢物和多肽进行了定量分析。同时,通过N-磷酰化氨基酸标记,建立了手性化合物分离新策略,基于普通反相色谱柱对手性胺、手性醇及手性小肽等代谢物及手性药物进行了成功分离与分析。
参考文献:
1.Guo,K.;Ji,C.J.;Li,L.Stable-isotope dimethylation labeling combinedwith LC-ESI MS for quantification of amine-containing metabolites inbiological samples.Anal.Chem.2007,79,8631-8638.
2.Li,S.F.;Jin,Y.B.;Tang,Z.;Lin,S.H.;Liu,H.X.;Jiang,Y.Y.;Cai,Z.W.Anovel method of liquid chromatography-tandem mass spectrometry combined withchemical derivatization for the determination of ribonucleosides inurine.Anal.Chim.Acta 2015,864,30-38.
3.Zhou,R.K.;Guo,K.;Li,L.5-Diethylamino-naphthalene-1-sulfonylchloride(DensCl):A novel triplex isotope labeling reagents for quantitativemetabolome analysis by liquid chromatography massspectrometry.Anal.Chem.2013,85,11532-11539.
4.Gao,X.;Wu,H.Z.;Lee,K.C.;Liu,H.X.;Zhao,Y.F.;Cai,Z.W.;Jiang,Y.Y.Stable isotope N-phosphorylation labeling for peptide de novo sequencingand protein quantification based on organic phosphoruschemistry.Anal.Chem.2012,84,10236-10244.
5.Gao,X.;Tang,Z.;Lu,M.H.;Liu,H.X.;Jiang,Y.Y.;Zhao,Y.F.;Cai,Z.W.Suppression of matrix ions by N-phosphorylation labeling using matrix-assisted laser desorption-ionization time-of-flight massspectrometry.Chem.Commun.2012,48,10198–10200.
发明内容
本发明的目的在于提供一种稳定同位素标记手性磷试剂及其制备方法。
本发明的另一目的在于提供所述稳定同位素标记手性磷试剂在对含有氨基手性化合物的手性比例进行定量分析;对含有氨基代谢物,如氨基酸、小肽或多肽等进行稳定同位素磷试剂标记定量质谱分析;对生物样品中含有氨基的代谢物进行定量质谱分析中的应用。
所述稳定同位素标记手性磷试剂的结构式如下:
其中,a为16或18;b为12或13;c为1或2;d为14或15;n为1、2、3、4或5,首选1、2和3;R为L-或D-氨基酸的侧链取代基(A)-(H),首选(A)、(B)和(G),包括如下结构:
A为N-羟基化合物取代基,如N-羟基琥珀酰亚胺(I)、N-羟基邻苯二甲酰亚胺(J)、N-羟基戊二酰亚胺(K)、N-羟基-5-降冰片烯-2,3-二甲酰亚胺(L)、1-羟基苯并三氮唑(M)及3-羟基-4-氧-3,4-二氢-1,2,3-苯并三氮嗪(N)等,首选N-羟基琥珀酰亚胺(I)及1-羟基苯并三氮唑(M),化学结构如下:
所述稳定同位素标记手性磷试剂的制备方法,包括以下步骤:
步骤1:稳定同位素标记的醇1的制备:
在10~60℃的温度范围内,卤代烷烃与水在含有银盐与有机溶剂的混合体系中搅拌反应,反应6~72h后可以得到产物1。延长反应时间对反应没有影响。
所述卤代烷烃可采用含C1~C6的直链或支链的氯代、溴代或碘代烷烃,优选溴代或碘代的烷烃。
所述卤代烃中的C是碳-12或碳-13,H是氢-1或氢-2。
所述水中的O是氧-16或氧-18。
所述银盐是无机酸的银盐或含C1~C6的羧酸的银盐,优选醋酸银、四氟硼酸银或硝酸银。
所述有机溶剂是醚类溶剂,优选乙醚或四氢呋喃。
步骤2:稳定同位素标记的亚磷酸酯2的制备:
产物1与亚磷酸二苯酯按摩尔比2︰1在有机溶剂中反应,反应中加入0.1~10当量的碱,反应时间为1~12h,反应完后浓缩除去有机溶剂,经柱层析分析得到产物亚磷酸酯2。
所述亚磷酸二苯酯中的O为氧-16或氧-18。
所述碱可以是无机碱或有机碱,如氢氧化钠、氢氧化钾、吡啶或三乙胺等,优选吡啶或三乙胺。
所述有机溶剂是醚类溶剂,如乙醚、四氢呋喃或1,4-二氧六环等,优选乙醚或四氢呋喃。
步骤3:稳定同位素标记的N-磷酰化氨基酸3的制备:
冰浴条件下,2与四氯化碳按摩尔比1︰2混合加入氨基酸水-有机溶剂混合溶液中,反应中加入0.1~10当量的碱,反应2~4h后升至室温,再反应1~12h,反应完成后浓缩除去有机溶剂,用盐酸溶液调节pH=2~4,乙酸乙酯萃取,浓缩残余产物重结晶得到N-磷酰化氨基酸3。
所述氨基酸可采用D-或L-氨基酸,其中C为碳-12或碳-13;O为氧-16或氧-18;N为氮-14或氮-15。
所述碱是有机碱,如吡啶、三乙胺或三丁胺等,优选吡啶或三乙胺。
所述有机溶剂是乙醇、甲醇、丙酮或乙腈等,优选乙腈或乙醇。
步骤4:稳定同位素标记N-磷酰化氨基酸活化酯4的制备:
3与N-羟基化合物按摩尔比1︰1.2在有机溶剂中反应,反应中加入1.5当量的脱水缩合试剂,反应1~12h,反应完后过滤除去沉淀,浓缩除去有机溶剂,重结晶得到N-磷酰化氨基酸活化酯4。其中,A为N-羟基化合物,包括N-羟基琥珀酰亚胺(I)、N-羟基邻苯二甲酰亚胺(J)、N-羟基戊二酰亚胺(K)、N-羟基-5-降冰片烯-2,3-二甲酰亚胺(L)、1-羟基苯并三氮唑(M)及3-羟基-4-氧-3,4-二氢-1,2,3-苯并三氮嗪(N)等,首选N-羟基琥珀酰亚胺(I)及1-羟基苯并三氮唑(M)。
所述有机溶剂可为二氯甲烷、乙酸乙酯或乙醚等,首选二氯甲烷。
所述缩合试剂可为二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,优选二环己基碳二亚胺。
本发明中,磷酰化氨基酸活化酯试剂4标记含有氨基化合物的方法如下:
常温条件下,4与氨基化合物按摩尔比1.2︰1在水或有机溶剂中反应,反应中加入0.1~10当量的碱,反应0.5h。
碱可以是无机碱或有机碱,无机碱是氢氧化钠、氢氧化钾、碳酸氢钠、碳酸氢钾、碳酸钠或碳酸钾,有机碱是吡啶或三乙胺等;在水中优选碳酸氢钠或碳酸氢钾,在有机溶剂中优选三乙胺。
有机溶剂是乙腈、二氯甲烷、乙醚、四氢呋喃或氯仿,优选乙腈。
所述稳定同位素标记手性磷试剂的应用包括:
(1)对含有氨基手性化合物的手性比例进行定量分析:基于核磁共振-磷谱分析技术(31P NMR),手性磷标记试剂衍生化后,对非对映异构体的磷谱峰面积进行积分,实现手性代谢物异构体手性定量分析;基于高效液相色谱技术,手性磷试剂衍生化后对非对映异构体进行拆分,通过色谱峰积分面积比例进行手性定量分析;
(2)对含有氨基代谢物,如氨基酸、小肽或多肽等,进行稳定同位素磷试剂标记定量质谱分析;
(3)对生物样品中含有氨基的代谢物进行定量质谱分析,如尿样、血样和细胞等。
该试剂可以对含有氨基的代谢物如氨基酸、小肽和多肽等生物小分子选择性标记,通过质谱进行定量分析;该试剂可以运用于生物样品中手性氨基代谢物的定性与定量分析,如尿液、血液及细胞等。
该标记方法基于核磁共振磷谱(31P NMR)、高效液相色谱及质谱分析检测技术。
本发明是一种含有碳-13、氧-18或氮-15标记的新型含磷手性标记试剂及其制备方法,并且提供这种标记试剂用于含有氨基官能团代谢物及肽段标记的方法。同时提供该试剂运用于含氨基代谢物标准品及生物样品中氨基代谢物质谱定量分析。
与其它标记的方法相比,本发明具有以下突出优点:
1、可以广泛应用与各种含氨基类的手性化合物的定量分析,实现手性异构体的定量分析;
2、标记试剂具有质谱增敏效应,同时标记有利于大极性代谢物的色谱分析,
3、能用核磁共振-磷谱技术(31P NMR)进行样品直接定量分析;
4、“一步衍生化”反应时间短(约15min),标记具有高效和高选择性,样品处理步骤简单。
附图说明
图1为稳定同位素含磷手性标记定量分析含氨基手性代谢物示意图。
图2为碳-12,氧-16标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记D/L-苯丙氨酸(D/L-Phe)的31P NMR分析叠加图。
图3为碳-12,氧-16标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记不同手性比例D/L-苯丙氨酸(D/L-Phe)的31P NMR定量线性图。
图4为碳-12,氧-16标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记10种D/L-混合氨基酸的手性拆分色谱图。
图5为碳-12,氧-16标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记D/L-苯丙氨酸(D/L-Phe)的色谱-紫外吸收(HPLC-UV)分析叠加图。
图6为碳-12,氧-16标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记D/L-苯丙氨酸(D/L-Phe)的色谱-紫外吸收(HPLC-UV)分析定量线性图。
图7为碳-12,氧-16/碳-12,氧-18标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记L-丙氨酸(L-Ala)的质谱分析叠加图。
图8为碳-12,氧-16/碳-12,氧-18标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯标记质谱定量分析L-丙氨酸(L-Ala)的线性关系图。
图9为标准样品二异丙氧基-N-磷酰化-L-丙氨酸-L-丙氨酸二肽的色谱-质谱最低检测限浓度分析图。
图10为标准样品二异丙氧基-N-磷酰化-L-丙氨酸-L-丙氨酸二肽的色谱-质谱定量分析线性范围图。
图11为稳定同位素手性磷标记试剂运用于人尿样中氨基酸定量的色谱-质谱图。
图12为稳定同位素手性磷标记试剂运用于人尿样中氨基酸定量的分析结果。
具体实施方式
以下实施例将结合附图对本发明做进一步说明。
实施例一、碳-12,氧-18标记-二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备
步骤1、氧-18同位素标记的异丙醇(1)的制备
在25mL的烧瓶中,氮气氛保护下,加入2.55g 2-碘丙烷(15mmol),2.8g硝酸银(16.5mmol),1.1mL氧-18标记的水(60mmol),以及10mL乙醚。室温下搅拌反应24h后,加入1.4g碳酸氢钠,0.175g氯化钠及适量的干燥剂无水硫酸镁干燥4h,过滤除去沉淀。得(1)的乙醚溶液。
步骤2:含氧-18同位素标记的亚磷酸酯(2)的制备
在25mL烧瓶中,氮气氛保护保护下,向(1)的乙醚溶液中加入2mL三乙胺以及1.64g亚磷酸二苯酯(7mmol),室温下搅拌12h后,除去溶剂,粗产品经硅胶柱层析分离得到产物(2)。
1H NMR(400MHz,CDCl3):δ=6.74(d,J=687.46Hz,1H),4.69-4.57(m,2H),1.27(m,12H)ppm.
13C NMR(101MHz,CDCl3):δ=70.79(d,J=5.77Hz),23.96(d,J=4.44Hz),23.77(d,J=4.90Hz)ppm.
31P NMR(162MHz,CDCl3):δ=4.33ppm.
ESI-MS(正离子模式):[M+H]+,m/z 171.0914。
步骤3:氧-18同位素标记的二异丙氧基-N-磷酰化-L-丙氨酸(3)的制备
冰浴条件下,将0.102g氧-18同位素标记的亚磷酸二异丙酯(2)(0.6mmol)与0.25mL的四氯化碳的混合液缓慢滴入L-丙氨酸0.054g(0.6mmol)、三乙胺0.413mL、水0.4mL和0.165mL无水乙醇的混合溶液中,反应1h后缓慢升至室温3h。浓缩除去部分有机溶剂,剩余液体在冰浴下调节pH=2~3,再用乙酸乙酯萃取,浓缩除去溶剂即可得到白色固体状的氧-18同位素标记的二异丙氧基-N-磷酰化-L-丙氨酸(3)。
1H NMR(400MHz,CDCl3):δ=11.18(br,1H),4.67-4.52(m,2H),3.85-3.76(m,1H),1.41(d,J=7.0Hz,3H),1.30-1.27(m,12H).
13C NMR(101MHz,CDCl3):δ=176.01(d,J=10.4Hz),71.62(d,J=5.7Hz),71.37(d,J=5.6Hz),49.87,23.86-23.73(m),20.94(d,J=2.9Hz).
31P NMR(162MHz,CDCl3):δ=5.78ppm.
ESI-MS:[M+H]+,m/z 258.1237。
步骤4:含氧-18同位素标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯(4)的制备
在25mL的圆底烧瓶中加入51.4mg氧-18同位素标记的二异丙氧基-N-磷酰化-L-丙氨酸(3)(0.2mmol),23.5mg N-羟基琥珀酰亚胺(0.204mmol),61.8mg二环己基碳二亚胺(0.3mg),4mL二氯甲烷,室温下搅拌反应24h,过滤除去有机溶剂即可得到(4)即含氧-18同位素标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯固体(4)。
1H NMR(400MHz,CDCl3):δ=4.60-4.51(m,2H),4.21-4.09(m,1H),2.79(s,4H),1.53(d,J=7.2Hz,3H),1.29-1.21(m,12H)ppm.
13C NMR(101MHz,CDCl3):δ=169.80(d,J=6.1Hz),168.68,71.74(d,J=5.8Hz),71.48(d,J=5.7Hz),48.65,25.68,23.90-23.72(m),21.40(d,J=5.1Hz)ppm.
31P NMR(162MHz,CDCl3):δ=3.91ppm.
ESI-MS:[M+H]+,m/z 355.1400。
实施例二、碳-12,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备
同实施例一,省去步骤1、2,从步骤3开始,仅将步骤3中的(2)的氧-18标记的亚磷酸二异丙酯换成氧-16的亚磷酸二异丙酯,得到氧-16标记的二异丙氧基-N-磷酰化L-丙氨酸(5)。再将(5)用于步骤4中得到氧-16的二异丙氧基-N-磷酰化-L-丙氨酸活化酯(6)。
1H NMR(400MHz,CDCl3):δ=8.99(s,1H),4.61-4.51(m,2H),3.84-3.76(m,1H),1.39(d,J=7.05Hz,3H),1.29-1.23(m,12H)ppm.
13C NMR(101MHz,CDCl3):δ=176.01(d,J=10.36Hz),71.62(d,J=5.68Hz),71.37(d,J=5.62Hz),49.87,25.52(s),23.87-23.73(m),20.94(d,J=2.86Hz)ppm.
31P NMR(162MHz,CDCl3):δ=5.89ppm.
ESI-MS:[M+H]+,m/z 254.1153。
1H NMR(400MHz,CDCl3):δ=4.65-4.53(m,2H),4.25-4.15(m,1H),2.81(s,4H),1.56(d,J=7.2Hz,3H),1.32-1.27(m,12H)ppm.
13C NMR(101MHz,CDCl3):δ=169.80(d,J=6.3Hz),168.68,71.75(d,J=5.8Hz),71.50(d,J=5.7Hz),48.70,25.69,23.89-23.72(m),21.36(d,J=5.1Hz)ppm.
31P NMR(162MHz,CDCl3,ppm):δ=3.95ppm.
ESI-MS:[M+H]+,m/z 351.1323。
实施例三、碳-12,氧-18标记的二乙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1的2-碘丙烷换成碘乙烷。
实施例四、碳-12,氧-16标记的二乙氧基-N-磷酰-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,省去步骤1、2,从步骤3开始,仅将步骤3中的(2)的氧-18标记的亚磷酸二异丙酯换氧-16标记的亚磷酸二乙酯。
实施例五、碳-12,氧-18标记的二丁氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1的2-碘丙烷换成碘丁烷。
实施例六、碳-12,氧-16标记的二丁氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,省去步骤1、2,从步骤3开始,仅将步骤3中的(2)的氧-18标记的亚磷酸二异丙酯换成氧-16标记的亚磷酸二丁酯。
实施例七、碳-13,氧-18标记的二丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1中的2-碘丙烷换成碳-13标记的1-碘丙烷。
实施例八、碳-13,氧-16标记的二丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1中的2-碘丙烷换成碳-13标记的1-碘丙烷,氧-18标记的水换成氧-16标记的去离子水。
实施例九、碳-13,氧-18标记的二异丙氧基-N-磷酰化-L-丙氨-1-羟基苯并三氮唑活化酯的制备:
同实施例一,仅将步骤4中的N-羟基琥珀酰亚胺换成1-羟基苯并三氮唑。
实施例十、碳-13,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨基酸-1-羟基苯并三氮唑活化酯的制备:
同实施例一,仅将步骤1中氧-18标记的水换成氧-16标记的去离子水,步骤4中的N-羟基琥珀酰亚胺换成1-羟基苯并三氮唑。
实施例十一、碳-13,氧-18标记的二丁氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1中的2-碘丙烷换成碳-13标记的碘丁烷。
实施例十二、碳-13,氧-16标记的二丁氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的制备:
同实施例一,仅将步骤1中的2-碘丙烷换成碳-13标记的碘丁烷,氧-18标记的水换成氧-16标记的去离子水。
以下考察本发明的适用性:
以下色谱-质谱联用(HPLC-MS)实验所用仪器为:Thermo Scientific Q-ExactiveOrbitrap质谱仪,配备Open Accela自动进样器和Accela 1250蠕动泵(Thermo FisherScientific,Bremen,Germany);色谱条件为:流动相为乙腈(B)和醋酸铵水溶液(A),流动相洗脱梯度:0min,5%B;0~5min,5%B;5~35min,5%~50%B;35~36min,50%~90%B;36~40min,90%B;40~41min,90%~95%B。色谱柱:Waters XBridge C18column(5μm,2.1×150mm);质谱条件为:电喷雾温度300℃,喷雾电压3.5kV,毛细管温度360℃,S-lens RF值为35%,分辨率70000,分子量范围:m/z 50~1200。
1、核磁共振磷谱(31P NMR)定量分析手性异构体比例:
将D/L的苯丙氨酸分别按D︰L为1︰0.5、1︰1、1︰2、1︰4、1︰6、1︰8、1︰10、1︰20的比例混合于1M的碳酸氢钠水溶液中,其中氨基酸的浓度为100mM,各取上述混合液400μL,分别加入2.5当量的2M的碳-12,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的乙腈溶液,冰浴条件下反应5min,升至室温再反应10min。然后分别加入60μL的D2O再进行核磁共振P31CPD检测。结果显示,苯丙氨酸的两个手性异构体标记后的磷谱峰实现基线分离,且不同比例线性定量效果良好,如图2和图3。
2、色谱拆分定量效果考察:
碳-12,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯用于拆分10对手性氨基酸混合物:冰浴条件下,将50μL的50mM的活化酯的乙腈溶液滴入含有D/L-Ala、D/L-Val、D/L-Leu、D/L-Arg、D/L-His、D/L-Trp、D/L-Ser、D/L-Tyr、D/L-Phe和D/L-Lys各1mM的500μL的10mM碳酸氢钠溶液中,冰浴条件下反应5min,随后升至室温反应10min。反应完后用氮吹仪除去部分乙腈,冰浴条件下用1%的甲酸水溶液调节pH=3,固相萃取除盐后,浓缩除去溶剂,加入40%的乙腈水溶液500μL溶解。取10μL上述样品溶液用超纯水稀释50倍后进行HPLC-MS分离与分析。测试结果显示所有氨基酸在衍生化之后均与其对应异构体达到了较好的分离,均达到基线分离(Rs大于或等于1.5),如图4。
同上用碳-12,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯衍生D︰L为1︰0.5、1︰2、1︰4、1︰8、1︰12、1︰16、1︰20和1︰40的苯丙氨酸,其色谱累加图5及定量线性曲线为图6,R2达到0.9992,具有较好定量线性关系。
3、质谱定量分析效果考察:
分别用本发明的含磷衍生化试剂碳-12,氧-16标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯与碳-12,氧-18标记的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯分别衍生化L-Ala。其中氧-16:氧-18分别为0.5︰1、1︰1、2︰1、3︰1、5︰1、7︰1和10︰1。图7为质谱叠加图,图8为定量线性曲线。其中氨基酸的浓度及活化酯的用量以及衍生处理方法参照“2、色谱拆分定量效果考察”中的方法。
4、最低检测限考察:
分别为将标准品二异丙氧基-N-磷酰化-L-丙氨酸二肽用乙腈稀释成500nM~10pM不同浓度梯度溶液,随后进行质谱检测,结果显示在10pM时检测的信噪比大于3,定量线性曲线R2为1,证明该标记方法检测灵敏,定量效果很好,如图9和图10。
以下给出本发明应用于人的尿液样品中氨基酸及其他含有氨基代谢物的定量分析举例:
尿液处理:取2mL尿液于EP管中,12000转离心30min,取上层清液供标记用。
衍生化反应一:取500μL处理后的尿液,加入42毫克碳酸氢钠溶解。取24μL的50mM的碳-12,氧-18的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯乙腈溶液,冰浴下滴入上述500μL的尿液中,冰浴反应5min再升至室温反应10min。反应完成后氮吹仪除去部分乙腈,冰浴下用1%的甲酸水溶液调节pH=3。
衍生化反应二:冰浴条件下,将73μL的50mM的碳-12,氧-18的二异丙氧基-N-磷酰化-L-丙氨酸-N-羟基琥珀酰亚胺活化酯的乙腈溶液滴入含有混合氨基酸标准品,包括L-Asn、L-Pro、L-Asp、L-Glu、L-Gly、L-Ile、L-Met、L-Thr、L-Cys、L-Gln、D/L-Ala、D/L-Val、D/L-Leu、D/L-Arg、D/L-His、D/L-Trp、D/L-Ser、D/L-Tyr、D/L-Phe及D/L-Lys等各1mM的500μL的10mM碳酸氢钠溶液中,冰浴下反应5min,再升至室温反应10min,反应完成后,用氮吹仪除去部分乙腈,冰浴条件下用1%的甲酸水溶液调节pH=3。
将反应一、二调节完pH的反应液混合,固相萃取除盐,浓缩除去溶剂,加入40%的乙腈/水混合溶液(1︰1)500μL溶解。取10μL用超纯水稀释50倍后进行色谱-质谱分析。通过对比氧-16与氧-18标记的氨基酸质谱峰相对强度,对尿液中的氨基酸浓度进行绝对定量分析,定量结果如图11和12所示,20种L-氨基酸和10种D-氨基酸得到成功定量,其中L-氨基酸的浓度范围为0.45μM~1.12mM,D-氨基酸的浓度范围为0.2~25μM。
表1是本发明中所涉及的部分新型含磷手性标记试剂分子结构式。
表1
图1是基本发明的含有碳-13、氧-18或氮-15标记的新型含磷手性标记试剂衍生化方法原理示意图。
Claims (10)
1.一种稳定同位素标记手性磷试剂,其特征在于其结构式如下:
其中,a为16或18;b为12或13;c为1或2;d为14或15;n为1、2、3、4或5;R为L-或D-氨基酸的侧链取代基(A)-(H),包括如下结构:
A为N-羟基化合物取代基,如N-羟基琥珀酰亚胺(I)、N-羟基邻苯二甲酰亚胺(J)、N-羟基戊二酰亚胺(K)、N-羟基-5-降冰片烯-2,3-二甲酰亚胺(L)、1-羟基苯并三氮唑(M)及3-羟基-4-氧-3,4-二氢-1,2,3-苯并三氮嗪(N),化学结构如下:
2.如权利要求1所述一种稳定同位素标记手性磷试剂,其特征在于所述n为1、2和3;R为L-或D-氨基酸的侧链取代基(A)、(B)和(G);N-羟基化合物取代基为N-羟基琥珀酰亚胺(I)及1-羟基苯并三氮唑(M)。
3.如权利要求1所述稳定同位素标记手性磷试剂的制备方法,其特征在于包括以下步骤:
步骤1:稳定同位素标记的醇1的制备:
在10~60℃的温度范围内,卤代烷烃与水在含有银盐与有机溶剂的混合体系中搅拌反应,反应6~72h后可以得到产物1;
步骤2:稳定同位素标记的亚磷酸酯2的制备:
产物1与亚磷酸二苯酯按摩尔比2︰1在有机溶剂中反应,反应中加入0.1~10当量的碱,反应时间为1~12h,反应完后浓缩除去有机溶剂,经柱层析分析得到产物亚磷酸酯2;
步骤3:稳定同位素标记的N-磷酰化氨基酸3的制备:
冰浴条件下,2与四氯化碳按摩尔比1︰2混合加入氨基酸水-有机溶剂混合溶液中,反应中加入0.1~10当量的碱,反应2~4h后升至室温,再反应1~12h,反应完成后浓缩除去有机溶剂,用盐酸溶液调节pH=2~4,乙酸乙酯萃取,浓缩残余产物重结晶得到N-磷酰化氨基酸3;
步骤4:稳定同位素标记N-磷酰化氨基酸活化酯4的制备:
3与N-羟基化合物按摩尔比1︰1.2在有机溶剂中反应,反应中加入1.5当量的脱水缩合试剂,反应1~12h,反应完后过滤除去沉淀,浓缩除去有机溶剂,重结晶得到N-磷酰化氨基酸活化酯4;其中,A为N-羟基化合物,包括N-羟基琥珀酰亚胺(I)、N-羟基邻苯二甲酰亚胺(J)、N-羟基戊二酰亚胺(K)、N-羟基-5-降冰片烯-2,3-二甲酰亚胺(L)、1-羟基苯并三氮唑(M)及3-羟基-4-氧-3,4-二氢-1,2,3-苯并三氮嗪(N),首选N-羟基琥珀酰亚胺(I)及1-羟基苯并三氮唑(M)。
4.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤1中,所述卤代烷烃采用含C1~C6的直链或支链的氯代、溴代或碘代烷烃,优选溴代或碘代的烷烃。
5.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤1中,所述卤代烃中的C是碳-12或碳-13,H是氢-1或氢-2;
所述水中的O是氧-16或氧-18;
所述银盐是无机酸的银盐或含C1~C6的羧酸的银盐,优选醋酸银、四氟硼酸银或硝酸银;
所述有机溶剂是醚类溶剂,优选乙醚或四氢呋喃。
6.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤2中,所述亚磷酸二苯酯中的O为氧-16或氧-18;
所述碱可以是无机碱或有机碱,如氢氧化钠、氢氧化钾、吡啶或三乙胺,优选吡啶或三乙胺;
所述有机溶剂是醚类溶剂,如乙醚、四氢呋喃或1,4-二氧六环,优选乙醚或四氢呋喃。
7.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤3中,所述氨基酸采用D-或L-氨基酸,其中C为碳-12或碳-13;O为氧-16或氧-18;N为氮-14或氮-15;
所述碱是有机碱,如吡啶、三乙胺或三丁胺,优选吡啶或三乙胺;
所述有机溶剂是乙醇、甲醇、丙酮或乙腈,优选乙腈或乙醇。
8.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤4中,所述有机溶剂为二氯甲烷、乙酸乙酯或乙醚,首选二氯甲烷;
所述缩合试剂可为二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,优选二环己基碳二亚胺。
9.如权利要求3所述稳定同位素标记手性磷试剂的制备方法,其特征在于在步骤4中,磷酰化氨基酸活化酯试剂4标记含有氨基化合物的方法如下:
常温条件下,4与氨基化合物按摩尔比1.2︰1在水或有机溶剂中反应,反应中加入0.1~10当量的碱,反应0.5h;
碱可以是无机碱或有机碱,无机碱是氢氧化钠、氢氧化钾、碳酸氢钠、碳酸氢钾、碳酸钠或碳酸钾,有机碱是吡啶或三乙胺;在水中优选碳酸氢钠或碳酸氢钾,在有机溶剂中优选三乙胺;
有机溶剂是乙腈、二氯甲烷、乙醚、四氢呋喃或氯仿,优选乙腈。
10.如权利要求1所述稳定同位素标记手性磷试剂的应用,其特征在于包括:
(1)对含有氨基手性化合物的手性比例进行定量分析:基于核磁共振-磷谱分析技术(31PNMR),手性磷标记试剂衍生化后,对非对映异构体的磷谱峰面积进行积分,实现手性代谢物异构体手性定量分析;基于高效液相色谱技术,手性磷试剂衍生化后对非对映异构体进行拆分,通过色谱峰积分面积比例进行手性定量分析;
(2)对含有氨基代谢物,如氨基酸、小肽或多肽,进行稳定同位素磷试剂标记定量质谱分析;
(3)对生物样品中含有氨基的代谢物进行定量质谱分析,如尿样、血样和细胞。
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