CN113956321A - 雌二醇6位葡萄糖醛酸苷及其制备方法和应用 - Google Patents
雌二醇6位葡萄糖醛酸苷及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及有机合成技术领域,特别涉及雌二醇6位葡萄糖醛酸苷及其制备方法和应用。该化合物合成方法:首先将6‑酮‑雌二醇二乙酸酯催化加氢还原得到6‑羟基‑雌二醇二乙酸酯;所得化合物和2,3,4‑三‑O‑乙酰基‑1‑O‑三氯乙亚胺酰基‑吡喃葡萄糖甲酯反应得到2,3,4‑三‑O‑乙酰基‑1‑O‑雌二醇二乙酸甲酯基‑葡萄糖醛酸甲酯苷;所得化合物碱性条件下脱乙酰基得到1‑O‑雌二醇基‑葡萄糖醛酸甲酯苷;所得化合物脱保护和酸化得到目标化合物。该化合物的合成为首例公开,同时本发明化合物的合成工艺简单,收率高,且以该化合物为原料制备所得雌二醇检测试剂盒特异性强、精密性高、准确度高、发光值梯度和稳定性优良。
Description
技术领域
本发明涉及有机合成技术领域,特别涉及雌二醇6位葡萄糖醛酸苷及其制备方法和应用。
背景技术
雌二醇(Estradiol,E2)是一种甾体雌激素,男性由睾丸分泌,未孕女性由卵巢分泌。女性血清E2测定对评价各种月经异常是很有用的指标:如女孩青春期提前或延迟、原发性或继发性闭经、卵巢早衰等。同时,血清E2也可用于衡量卵巢卵泡的成熟度。怀孕的女性血清E2水平明显升高,远高于上述的排卵前峰值水平,而且高水平的E2浓度持续整个孕期。男性E2水平的升高常与女性化综合征、乳房女性化等疾病有关。在不孕症患者中,血清E2的监测对于监控诱导排卵及随后的治疗是非常有用的。在体外受精(IVF)中,对卵巢进行过刺激时,通常每天对绒毛膜促性腺激素的使用和卵母细胞的收集进行调整,也需要检测血清E2浓度。因此,检测人体生物样本中的雌二醇的含量具有重要的临床意义。
目前,雌二醇检测的常用方法主要有放射免疫分析法、酶联免疫分析法和化学发光免疫分析法等。放射免疫分析法合成工艺复杂,有效期短,存在放射性污染;酶联免疫分析法需进行手工操作,误差较大;化学发光免疫分析技术发展迅速,灵敏度、特异性以及自动化程度高。当前研究的主要方向是合成雌二醇衍生物,使用该化合物制备免疫原性强的雌二醇免疫原,进而制备可用于全自动化学发光仪的检测试剂。
目前雌二醇衍生物的制备暂无雌二醇6位葡萄糖醛酸苷的报道,更没有该化合物在雌二醇检测试剂上应用情况的报道。
发明内容
有鉴于此,本发明提供了雌二醇6位葡萄糖醛酸苷及其制备方法和应用。该化合物为新的雌二醇衍生物,以该化合物为原料制备所得雌二醇检测试剂特异性强、精密性高、准确度高、发光值梯度和稳定性优良。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种化合物,该化合物为雌二醇6位葡萄糖醛酸苷,化合物的结构式如式I所示:
本发明还提供了该化合物的制备方法,包括如下步骤:
步骤(1)在钯碳催化作用下,6-酮-雌二醇二乙酸酯与氢气发生催化加氢还原反应,得到6-羟基-雌二醇二乙酸酯;
步骤(2)在氮氛保护和三氟化硼乙醚催化作用下,2,3,4-三-O-乙酰基-1-O-三氯乙亚胺酰基-吡喃葡萄糖甲酯与6-羟基-雌二醇二乙酸酯发生取代反应,得到2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷;
步骤(3)在甲醇钠催化作用下,2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷发生脱乙酰基反应,得到1-O-雌二醇基-葡萄糖醛酸甲酯苷;
步骤(4)在碱性条件下,1-O-雌二醇基-葡萄糖醛酸甲酯苷发生醇解反应,再经酸化处理后,得到雌二醇6位葡萄糖醛酸苷。
作为优选,步骤(1)反应所用有机溶剂为甲醇、二甲基亚砜、四氢呋喃或乙醇。
作为优选,步骤(1)反应时间为4~10h。
作为优选,步骤(2)反应所用有机溶剂为二氯甲烷、三氯甲烷或丙酮。
作为优选,步骤(2)反应时间为2~5h。
作为优选,步骤(3)反应所用有机溶剂为甲醇。
作为优选,步骤(3)反应时间为0.5~2h。
作为优选,步骤(4)反应所用有机溶剂为甲醇,碱性条件所用碱性溶液为氢氧化钠或氢氧化钾。
作为优选,步骤(4)反应时间为20~60min。
本发明还提供了该化合物在制备雌二醇检测试剂中的应用。
本发明提供了雌二醇6位葡萄糖醛酸苷及其制备方法和应用。本发明具有的技术效果为:
本发明化合物的合成为首例公开雌二醇6位葡萄糖醛酸苷制备,同时本发明化合物的合成工艺简单,收率高,且以该化合物为原料制备所得雌二醇检测试剂特异性强、精密性高、准确度高、发光值梯度和稳定性优良。
附图说明
图1是本发明化合物的合成路线图。
具体实施方式
本发明公开了雌二醇6位葡萄糖醛酸苷及其制备方法和应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
以下实施例中所用中英文对照:
HRP:辣根过氧化物酶(HorseradishPeroxidase)
MES:吗啉乙磺酸(4-Morpholineethanesulfonic acid)
BSA:牛血清白蛋白
ADP:氨基比林(4-DiMethylaMino Antipyrine)
P300:Proclin 300防腐剂
DSS:葡聚糖硫酸钠(Dextran Sulfate Sodium Salt)
EDTA:乙二胺四乙酸
在本发明提供的具体实施例中,雌二醇6位葡萄糖醛酸苷制备方法包括下述具体步骤:
第一步,将化合物6-酮-雌二醇二乙酸酯加入有机溶剂溶解,然后加入钯碳有机溶液,氢气氛围下进行催化加氢还原,反应完成后,过滤收集滤液,减压浓缩除去溶剂,经柱层析纯化得到化合物6-羟基-雌二醇二乙酸酯,备用;
第二步,将2,3,4-三-O-乙酰基-1-O-三氯乙亚胺酰基-吡喃葡萄糖甲酯溶于有机溶剂中,加入6-羟基-雌二醇二乙酸酯,搅拌均匀,在氮氛保护下,加入三氟化硼乙醚,反应完全后,加入饱和碳酸氢钠溶液,萃取收集有机相,加入饱和食盐水,萃取收集有机相,加入无水硫酸镁干燥,减压浓缩除去溶剂,经柱层析纯化得到2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷,备用;
第三步,将第二步所得化合物在甲醇溶液中经甲醇钠催化反应,反应完成后经活性炭脱色吸附、过滤、柱层析纯化得到1-O-雌二醇基-葡萄糖醛酸甲酯苷,备用;
第四步,将第三步所得化合物溶解在甲醇溶液中,加入碱性溶液,反应完全后加入盐酸进行酸化处理,反应液加入活性炭脱色吸附、过滤、柱层析纯化得到目标衍生物雌二醇6位葡萄糖醛酸苷。
本发明中所用试剂或仪器均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1制备化合物雌二醇6位葡萄糖醛酸苷
其合成路线如图1所示,具体步骤为:
1、6-羟基-雌二醇二乙酸酯的制备
取化合物6-酮-雌二醇二乙酸酯3.7g于500mL圆底烧瓶中,加入100mL无水甲醇溶解,通氢气,逐滴加入含有0.37g钯碳的20mL甲醇溶液,搅拌反应5h后,过滤收集滤液,减压浓缩除去溶剂,粗产品经柱层析纯化得到化合物6-羟基-雌二醇二乙酸酯白色粉末3.3g,收率为88.7%。MS(ESI):m/z(%)395.2[M+Na]+。
2、2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷的制备
将2,3,4-三-O-乙酰基-1-O-三氯乙亚胺酰基-吡喃葡萄糖甲酯4.8g置于250mL圆底烧瓶中,加入100mL二氯甲烷搅拌溶解,加入1.9g 6-羟基-雌二醇二乙酸酯,搅拌均匀,在氮氛保护下,加入378μL三氟化硼乙醚,反应3h后,加入50mL饱和碳酸氢钠溶液,萃取收集有机相,有机相加入饱和食盐水,萃取收集有机相,加入5g无水硫酸镁干燥,减压浓缩除去溶剂,经柱层析纯化得到2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷白色固体粉末3.1g,收率88.2%。MS(ESI):m/z(%)711.3[M+Na]+。
3、1-O-雌二醇基-葡萄糖醛酸甲酯苷制备
将以上所得化合物2.0g置于250mL烧瓶中,加入100mL甲醇搅拌溶解,加入甲醇钠0.16g,反应1h后,加入0.1g活性炭脱色吸附,过滤除去活性炭及不溶杂质,柱层析纯化得到1-O-雌二醇基-葡萄糖醛酸甲酯苷1.2g,收率86.4%。MS(ESI):m/z(%)501.2[M+Na]+。
4、雌二醇6位葡萄糖醛酸苷的制备
取以上所得化合物0.5g于100mL圆底烧瓶中,加入20mL甲醇搅拌均匀,加入1mL浓度为1mol/L氢氧化钠溶液,反应30min后,加入盐酸酸化至中性,反应液加入0.05g活性炭脱色吸附,过滤,柱层析纯化得到目标衍生物雌二醇6位葡萄糖醛酸苷0.4g,收率82.5%。MS(ESI):m/z(%)487.2[M+Na]+。1HNMR(400MHz,CDCl3):δ(ppm)12.6(s,1H,COOH),9.32(s,1H,O-H),6.73(d,1H,Ar-H),6.62(d,1H,Ar-H),6.42(d,1H,Ar-H),5.43(d,1H,CH),4.45-4.79(m,5H),4.03(t,1H,CH),3.89(t,1H,CH),3.69(t,1H,CH),3.42(t,1H,CH),2.66(q,1H,CH),1.31-2.00(m,10H),1.30(d,1H,CH),1.04(q,1H,CH),0.94(s,3H,CH3)。
实施例2应用测试试验
利用雌二醇检测试剂盒(磁微粒化学发光法)结合制备的化合物进行应用测试,具体如下:
所采用的雌二醇检测试剂盒(磁微粒化学发光法)包括:雌二醇抗体包被的磁微粒悬浮液;HRP标记雌二醇制备酶结合物;免疫反应形成抗体-酶标抗原复合物。其中本发明涉及磁微粒悬浮液配方:pH6.0MES+0.5%BSA+3‰ADP+0.5‰P300+1.5%NaCl+0.75%DSS+400ng/mL甲基睾酮+1.5%EDTA+0.5%乙醇;酶结合物配方:pH6.0MES+0.5%BSA+3‰ADP+0.5‰P300+1.5%NaCl。
实施例1化合物雌二醇6位葡萄糖醛酸苷与郑州安图生物工程股份有限公司在产试剂盒所用3位雌二醇衍生物(对照)分别标记HRP,加入上述酶结合物中(酶结合物浓度为1/1K)。
上述对照所用3位雌二醇衍生物结构式为:
1、梯度测试
将购于Sigma公司雌二醇溶解于无水甲醇中,配制成1μg/mL的溶液,将溶液稀释于不含雌二醇的空白人造血清中,至浓度分别为4500、1500、500、100、30、0pg/mL,将上述溶液作为校准品进行ELISA实验,结果如表1所示。
表1
由表1结果可知,采用本发明化合物所配制试剂盒校准品梯度S0/S1和S0/S5明显优于对照,发光值梯度优良。
2、精密性测试
将配制好的S0-S5六个雌二醇校准品,分别用实施例1和上述对照化合物结合雌二醇检测试剂盒(磁微粒化学发光法)重复测试30次,统计数据精密度(CV%),结果如表2所示。
表2
由表2结果可知,采用本发明化合物所配制试剂盒S0-S5精密度明显优于对照,精密性高。
3、药物与激素干扰试验
选取常见药物、激素及激素代谢物进行干扰试验,调整浓度为1ng/mL,采用实施例1和对照化合物结合雌二醇检测试剂盒分别测定上述干扰物,结果如表3所示。
表3
由表3中结果可知,实施例1化合物所制备雌二醇检测试剂盒干扰试验明显优于对照,且与常见干扰物无交叉反应,特异性强。
4、相关性分析
分别采用实施例1和对照衍生物制备雌二醇检测试剂盒进行200份临床样本的测试,同时对样本进行高效液相色谱法测试,对数据进行作图分析,得出相关系数R2。结果如表4所示。
表4
对照 | 实施例1 | |
相关系数R2 | 0.9121 | 0.9982 |
由表4结果可知,本发明所得化合物制备雌二醇检测试剂测定雌二醇临床样本的准确度高。
5、稳定性评价
将实施例1和对照应用于雌二醇检测试剂盒(磁微粒化学发光法),所得试剂盒置于37℃,分别放置0、3、5、7和10天后进行项目评价验证,统计发光值降幅情况,结果如表5所示。
表5
由表5结果可知,本发明所得化合物制备雌二醇检测试剂稳定性明显优于对照,稳定性良好。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
2.权利要求1所述化合物的制备方法,其特征在于,包括如下步骤:
步骤(1)在钯碳催化作用下,6-酮-雌二醇二乙酸酯与氢气发生催化加氢还原反应,得到6-羟基-雌二醇二乙酸酯;
步骤(2)在氮氛保护和三氟化硼乙醚催化作用下,2,3,4-三-O-乙酰基-1-O-三氯乙亚胺酰基-吡喃葡萄糖甲酯与6-羟基-雌二醇二乙酸酯发生取代反应,得到2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷;
步骤(3)在甲醇钠催化作用下,2,3,4-三-O-乙酰基-1-O-雌二醇二乙酸甲酯基-葡萄糖醛酸甲酯苷发生脱乙酰基反应,得到1-O-雌二醇基-葡萄糖醛酸甲酯苷;
步骤(4)在碱性条件下,1-O-雌二醇基-葡萄糖醛酸甲酯苷发生醇解反应,再经酸化处理后,得到雌二醇6位葡萄糖醛酸苷。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)反应所用有机溶剂为甲醇、二甲基亚砜、四氢呋喃或乙醇。
4.根据权利要求2所述的制备方法,其特征在于,步骤(1)反应时间为4~10h。
5.根据权利要求2所述的制备方法,其特征在于,步骤(2)反应所用有机溶剂为二氯甲烷、三氯甲烷或丙酮。
6.根据权利要求2所述的制备方法,其特征在于,步骤(2)反应时间为2~5h。
7.根据权利要求2所述的制备方法,其特征在于,步骤(3)反应所用有机溶剂为甲醇。
8.根据权利要求2所述的制备方法,其特征在于,步骤(3)反应时间为0.5~2h。
9.根据权利要求2至8中任一项所述的制备方法,其特征在于,步骤(4)反应所用有机溶剂为甲醇,所述碱性条件所用碱性溶液为氢氧化钠或氢氧化钾;
步骤(4)反应时间为20~60min。
10.权利要求1所述化合物在制备雌二醇检测试剂中的应用。
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