CN116082420B - 一种eps-g7的化学合成方法 - Google Patents
一种eps-g7的化学合成方法 Download PDFInfo
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Abstract
本发明提供一种EPS‑G7的化学合成方法，通过将β‑环糊精A乙酰化得到β‑乙酰环糊精B，B在乙酸酐/浓硫酸的混合液中开环，得到链状乙酰化七糖C，使用三氟化硼乙腈将对硝基苯酚与中间体C缩合，得到以α为优势构型的关键中间体p‑硝基苯基(G1)‑O‑乙酰基‑D‑麦芽七糖苷D，D在甲醇钠的作用下，将乙酰基脱除，得到pNP‑G7(p‑硝基苯基(G1)‑D‑麦芽七糖苷)，pNP‑G7在对甲苯磺酸和二甲氧基乙烷的存在下，末端单糖的C4和C6上的羟基之间形成缩醛保护，得到EPS‑G7。本发明方法所用的反应原料廉价易得，实现了α‑淀粉酶检测试剂EPS‑G7的化学合成，是一种简捷、经济、高效的合成方法。
Description
技术领域
本发明属于有机合成技术领域，涉及α-淀粉酶检测试剂的合成方法，具体涉及一种EPS-G7的化学合成方法。
背景技术
α-淀粉酶在人体生理循环中起着关键作用，被用作一些相关疾病的重要指标，如急性胰腺炎、急性有机磷农药中毒、焦虑或抑郁等。
EPS-G7作为α-淀粉酶的底物，是一种体外检测人血清或尿液中α-淀粉酶(AMS)活性的商用检测试剂，具有灵敏度高、抗干扰能力强的优势。EPS-G7的化学名为4,6-二甲氧基乙烷-(G7)-对硝基苯(G1)-α-D-麦芽七糖苷，其分子量为1299.41，是白色或近白色固体粉末，结构式如下。
EPS-G7由对硝基苯(G1)-α-D-麦芽七糖苷(pNP-α-G7)经一步化学修饰得到，因此pNP-α-G7的制备是合成EPS-G7的关键。到目前为止，文献中pNP-α-G7都是通过酶法制备，最常见的方法有两种。第一种是将α-环糊精开环得到麦芽六糖，再与对硝基苯-α-D-葡萄糖苷(pNP-α-G1)在酶的催化下合成pNP-α-G7。第二种是转糖基化法，由pNP-α-G1和麦芽七糖在转移酶催化下制备pNP-α-G7。
化学合成EPS-G7的关键在于pNP-α-G7的α-选择性合成。而目前糖基化反应的方法大多生成β-糖苷，α-糖苷，特别是1,2-顺式-α-糖苷的化学合成较难实现立体选择性，且现有的方法存在底物活化步骤长、底物受限、需要金属催化剂参与等不足。因此，开发新型合成方法实现1,2-顺式-α-糖苷键的构建并应用于EPS-G7的化学合成具有重要的应用价值。
发明内容
本发明的目的是提供一种EPS-G7的化学合成方法，合成路线如下：
具体通过以下步骤实现：
(1)β-环糊精A与醋酐在20-30℃下反应12h，得到β-乙酰环糊精B；
(2)将中间体B溶于乙酸酐/浓硫酸(V/V＝49:1)的混合液中，在40-60℃下反应24h，得到链状乙酰化七糖C；
(3)在缩合试剂存在的条件下，中间体C与对硝基苯酚在0-40℃反应1-9h，得到以α为优势构型的关键中间体p-硝基苯基(G1)-O-乙酰基-D-麦芽七糖苷D；
(4)关键中间体D与甲醇钠在20-30℃下反应24-48h，得到pNP-α-G7(p-硝基苯基(G1)-D-麦芽七糖苷)；
(5)pNP-α-G7在对甲苯磺酸和二甲氧基乙烷的存在下，在55℃反应24h，得到EPS-G7(4,6-亚乙基-(G7)-p-硝基苯基(G1)-α-D-麦芽七糖苷)。
其中，步骤(3)中的缩合试剂可以为三氟化硼乙腈、三氟化硼乙醚或四氯化锡等，优选三氟化硼乙腈。
步骤(3)中的缩合试剂用量可以为1-4当量，优选3当量。
步骤(3)中的反应溶剂可以为二氯甲烷、乙腈、甲苯或1,2-二氯乙烷等，优选二氯甲烷。
步骤(3)中的反应温度可以为0-40℃，优选25-30℃。
步骤(3)中的反应时间可以为1-9h，优选4-5h。
本发明提供的合成方法是以β-环糊精为起始原料，依次经过乙酰化、水解开环、缩合、酯水解和缩醛保护五步反应，反应条件温和，所用的反应原料廉价易得，为EPS-G7的化学合成提供了一种简单易行的方法。本发明所述的化学合成方法未见文献报道，且相比于酶法合成，此方法更加经济、高效。
本发明提供的合成路线的关键在于设计了基于三氟化硼乙腈配位的乙酰化七糖与对硝基苯酚的缩合反应，α/β比例可达80:20。此反应避免了繁琐的糖基供体或糖基受体预活化过程，在室温条件下即可进行，试剂价格低廉，后处理操作简单，为寡糖衍生物的合成和糖基化反应提供了简便的方法。
具体实施方式
下面结合具体的实施例对发明内容作进一步解释和说明，但是它们并不构成对本发明范围的限制或限定。
实施例1乙酰化反应
将28gβ-环糊精A溶于50mL吡啶中，降温到0℃，加入60mL醋酐和2.8g DMAP，室温下(20-30℃)搅拌12h。原料反应完后将反应液倒入1000mL水中，用乙酸乙酯(200mL×3)萃取，合并有机相，依次用盐酸(500mL×2,1M)、饱和碳酸氢钠(500mL×2)和饱和氯化钠(500mL)洗涤，经无水硫酸钠干燥后过滤，浓缩，得到50gβ-乙酰环糊精B。白色粉末；收率99％；M.p.235.1-236.4℃；1H NMR(500MHz,CDCl3)δ5.31(dd,J＝9.7,8.1Hz,7H),5.09(d,J＝3.8Hz,7H),4.83-4.77(m,7H),4.56(dd,J＝12.6,2.0Hz,7H),4.28(dd,J＝12.6,4.2Hz,7H),4.15(ddd,J＝9.8,4.2,1.9Hz,7H),3.71(dd,J＝9.6,8.0Hz,7H),2.13(s,21H),2.10(s,21H),2.06(s,21H)；13C NMR(125MHz,CDCl3)δ170.8(7C),170.5(7C),169.5(7C),96.8(7C),77.2(7C),70.8(7C),70.4(7C),69.6(7C),62.5(7C),20.8(7C),20.8(14C)。
实施例2糖苷键水解
将50gβ-乙酰环糊精B溶于100mL乙酸酐/浓硫酸(V/V＝49:1)的混合液中，于50℃搅拌24h，将所得混合物倒入1000mL水中，用乙酸乙酯(200mL×3)萃取，合并有机层，并依次用饱和碳酸氢钠(500mL×2)和饱和氯化钠(500mL×2)洗涤。经无水硫酸钠干燥后过滤，浓缩，得到的粗品用乙醇重结晶三次，得到28.2g中间体C。白色粉末；收率：54％；M.p.141.8-143.2℃；1H NMR(500MHz,CDCl3)δ6.23(d,J＝3.7Hz,1H),5.50(t,J＝9.5Hz,1H),5.42-5.27(m,12H),5.06(t,J＝9.9Hz,1H),4.94(dd,J＝10.0,3.8Hz,1H),4.84(dd,J＝10.5,4.1Hz,1H),4.72(dt,J＝9.9,4.5Hz,5H),4.58-4.44(m,6H),4.36-4.09(m,8H),4.04(dd,J＝10.4,6.1Hz,2H),4.00-3.84(m,11H),2.22(s,3H),2.20(s,9H),2.18(s,6H),2.14(s,3H),2.09(s,3H),2.06-2.00(m,21H),1.99(s,12H),1.98-1.95(m,12H)；13C NMR(125MHz,CDCl3)δ170.8(3C),170.7(3C),170.6,170.5,170.5(5C),169.9(2C),169.8,169.8,169.6(2C),169.5(3C),169.0,95.9,95.7,95.7(2C),95.6(2C),88.9,73.2(4C),72.3,72.2,71.7(5C),70.5(5C),70.2,70.0,69.8(2C),69.3,69.1,68.9(3C),68.4,67.9(2C),62.3(4C),62.1(2C),61.3,21.0,20.9(3C),20.9(5C),20.8(4C),20.8(2C),20.7,20.6(3C),20.5(3C),20.4。
实施例3缩合反应
将3.18g中间体C、2.8g对硝基苯酚混合于6mL二氯甲烷中，溶清后加入0.486g三氟化硼乙腈固体，25-30℃搅拌4h。反应结束后，往反应液中加入20mL水淬灭反应，搅拌，分层，有机层再依次用饱和碳酸氢钠和饱和氯化钠洗涤。经无水硫酸钠干燥后过滤，浓缩，得到的粗产品经硅胶柱层析(石油醚：乙酸乙酯＝2:3)分离得到1.43g中间体D-α和D-β的混合物(α/β＝80:20)。白色粉末；收率：43％；M.p.135.9-137.6℃；1H NMR(500MHz,CDCl3)δ8.23(d,J＝9.3Hz,2H),7.24(d,J＝9.2Hz,2H),5.76-5.66(m,1H),5.42-5.24(m,13H),5.05(t,J＝9.9Hz,1H),4.93(dd,J＝10.2,3.6Hz,1H),4.84(dd,J＝10.5,4.0Hz,1H),4.75-4.66(m,5H),4.54-4.43(m,6H),4.26(dddd,J＝16.4,10.0,6.9,3.5Hz,5H),4.20(dd,J＝12.3,2.5Hz,1H),4.15(dd,J＝12.3,2.6Hz,1H),4.08-4.00(m,3H),3.95-3.86(m,11H),2.20-2.16(m,12H),2.13(s,6H),2.08(s,3H),2.05-2.03(m,12H),2.02-2.00(m,9H),1.99-1.98(m,15H),1.97-1.93(m,9H)；13CNMR(125MHz,CDCl3)δ170.8,170.7(2C),170.7(2C),170.6,170.5,170.5,170.5,170.4(2C),170.3(2C),170.2,169.8(2C),169.6(2C),169.6(2C),169.5,169.5,160.7,143.2,125.9(2C),116.7(2C),95.9,95.7,95.7(2C),95.6(2C),94.1,73.2(4C),72.3,72.1(2C),71.7(3C),71.6(2C),70.5(3C),70.5(3C),70.1(2C),69.4(2C),69.2(2C),68.9(4C),68.4,67.9,62.3(3C),62.1,61.4,20.9,20.9(4C),20.9(3C),20.8(2C),20.8,20.7(2C),20.6(2C),20.6(3C),20.5(2C),20.5(2C)。
实施例4缩合反应
将3g中间体C、2.7g对硝基苯酚混合于6mL二氯甲烷中，溶清后加入0.47mL三氟化硼乙醚，25-30℃搅拌60-72h。往反应液中加入20mL水淬灭反应，搅拌，分层，有机层再依次用饱和碳酸氢钠和饱和氯化钠洗涤。经无水硫酸钠干燥后过滤，浓缩，得到的粗产品经硅胶柱层析(石油醚：乙酸乙酯＝2:3)分离得到1.2g中间体D-α和D-β的混合物(α/β＝75:25)，收率38％。
实施例5缩合反应
将3g中间体C、2.7g对硝基苯酚混合于6mL二氯甲烷中，溶清后加入0.5mL四氯化锡，25-30℃搅拌12-20h。往反应液中加入20mL水淬灭反应，搅拌，分层，有机层再依次用饱和碳酸氢钠和饱和氯化钠洗涤。经无水硫酸钠干燥后过滤，浓缩，得到的粗产品经硅胶柱层析(石油醚：乙酸乙酯＝2:3)分离得到1.26g中间体D-α和D-β的混合物(α/β＝60:40)，收率40％。
实施例6酯水解反应
将1g中间体D(α/β＝80:20)溶于10mL DCM/MeOH(V/V＝1:4)的混合液中，降温至0℃后，分批加入0.2g甲醇钠，在室温下(20-30℃)搅拌24-48h，反应完全后，过滤反应液，滤饼用DCM洗涤并烘干，得到0.52g淡黄色固体pNP-G7，收率90％。
实施例7缩醛保护
将0.52g pNP-G7溶于10mL DMF中，在溶液中加入0.2g对甲苯磺酸和0.28g二甲氧基乙烷，在55℃下搅拌24h。将反应液倒入100mL乙酸乙酯中析出产物，过滤，用乙酸乙酯洗涤滤饼，滤饼再通过反相柱层析(甲醇/水,20:80)分离纯化，得到0.313g的白色固体终产物EPS-G7，收率59％。M.p.213.6-214.8℃；[α]D25+126.5(c 0.55,CHCl3)；IR(KBr):1594,1345,1026,870cm-1；1H NMR(500MHz,DMSO-d6)δ8.22(d,J＝9.2Hz,2H),7.29(d,J＝9.3Hz,2H),5.65(d,J＝3.6Hz,1H),5.61-5.49(m,7H),5.44(dt,J＝8.0,3.5Hz,4H),5.41(d,J＝3.3Hz,1H),5.37(d,J＝6.4Hz,1H),5.24(d,J＝5.3Hz,1H),5.05(ddd,J＝14.2,9.8,4.1Hz,6H),4.68(q,J＝5.0Hz,1H),4.63(t,J＝5.6Hz,1H),4.59-4.50(m,5H),3.90(td,J＝9.3,8.8,3.7Hz,2H),3.70-3.38(m,29H),3.34-3.28(m,10H),3.10(t,J＝9.4Hz,1H),1.20(d,J＝5.0Hz,3H)；13C NMR(125MHz,DMSO-d6)δ162.1,141.7,125.8(2C),117.0(2C),101.1,100.3(2C),100.2(2C),98.6,97.4,80.4,79.2(3C),79.1,79.0(2C),73.2,73.1(5C),73.0,72.7,72.4,72.0(5C),71.6(5C),70.9 70.0,67.5,63.2,60.3(5C),60.0,19.9；HRMS(ESI)m/z calcd for C50H77O38Na[M+Na]+1322.4021,found 1322.4029。
Claims (9)
1.一种EPS-G7的化学合成方法，其特征在于，通过以下步骤实现：
（1）β-环糊精A与醋酐在20-30℃下反应12 h，得到 β-乙酰环糊精B；
（2）将中间体B溶于体积比为49:1的乙酸酐/浓硫酸混合液中，在40-60℃下反应24 h，得到链状乙酰化七糖C；
（3）在缩合试剂存在的条件下，中间体C与对硝基苯酚在0-40℃反应1-9 h，得到以α为优势构型的关键中间体p-硝基苯基(G1)-O-乙酰基-D-麦芽七糖苷D；
（4）关键中间体D与甲醇钠在20-30℃下反应24-48 h，得到pNP-α-G7（p-硝基苯基(G1)-D-麦芽七糖苷）；
（5）pNP-α-G7在对甲苯磺酸和二甲氧基乙烷的存在下，在55℃反应24 h，得到EPS-G7（4,6-亚乙基-(G7)-p-硝基苯基(G1)-α-D-麦芽七糖苷）；
合成路线如下：
。
2.根据权利要求1所述的化学合成方法，其特征在于，步骤（3）中的缩合试剂选择三氟化硼乙腈、三氟化硼乙醚或四氯化锡。
3.根据权利要求2所述的化学合成方法，其特征在于，步骤（3）中的缩合试剂选择三氟化硼乙腈。
4.根据权利要求1所述的化学合成方法，其特征在于，步骤（3）中的缩合试剂用量选择1-4当量。
5.根据权利要求4所述的化学合成方法，其特征在于，步骤（3）中的缩合试剂用量选择3当量。
6.根据权利要求1所述的合成方法，其特征在于，步骤（3）中的反应溶剂选用二氯甲烷、乙腈、甲苯或1,2-二氯乙烷。
7.根据权利要求6所述的化学合成方法，其特征在于，反应溶剂选用二氯甲烷。
8.根据权利要求1所述的化学合成方法，其特征在于，步骤（3）中的反应温度选择25-30℃。
9.根据权利要求1所述的化学合成方法，其特征在于，步骤（3）中的反应时间选择4-5h。
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