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CN106432371A - β‑1,2‑D‑寡聚甘露糖蛋白缀合物及其制备方法和应用 - Google Patents

β‑1,2‑D‑寡聚甘露糖蛋白缀合物及其制备方法和应用 Download PDF

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CN106432371A
CN106432371A CN201610928843.7A CN201610928843A CN106432371A CN 106432371 A CN106432371 A CN 106432371A CN 201610928843 A CN201610928843 A CN 201610928843A CN 106432371 A CN106432371 A CN 106432371A
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廖俊
何大为
刘超
吴也
付奔
潘炜华
吴秋业
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Second Military Medical University SMMU
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Abstract

本发明公开了一种如式I所示的β‑1,2‑D‑寡聚甘露糖蛋白缀合物及其制备方法和应用，式中n为0、1或2，R为载体蛋白KLH或HSA。本发明以α‑D‑葡萄糖为原料，合成了磷酸化的β‑1,2‑D‑寡聚甘露寡糖，并将其与载体蛋白偶联得到糖蛋白缀合物。将制备的化合物作为抗白念珠菌疫苗免疫小鼠，结果表明本发明的化合物能诱导机体产生较强的免疫应答，其中β‑1,2‑D‑甘露三糖蛋白缀合物的免疫原性最强且产生的免疫血清能够特异性识别白念珠菌细胞表面抗原，从而可以起到预防白念珠菌感染的目的，本发明的β‑1,2‑D‑寡聚甘露糖蛋白缀合物具有较强的应用价值。

Description

β-1,2-D-寡聚甘露糖蛋白缀合物及其制备方法和应用
技术领域
本发明涉及抗真菌糖疫苗技术领域，具体涉及β-1,2-D-寡聚甘露糖蛋白缀合物及其制备方法，该类糖蛋白缀合物作为疫苗在白念珠菌感染的预防和治疗中的应用。
背景技术
近年来，白念珠菌感染引起的死亡率不断上升，且病菌针对抗菌药物产生严重的耐药性，临床上需要有效的预防和治疗白念珠菌感染的药物和策略。真菌感染的免疫治疗主要采取的是主动性免疫治疗，即利用抗原刺激机体产生强大的免疫应答，通过产生抗体或分化效应细胞达到清除感染的真菌或其它微生物的目的，具有毒副作用小、疗效显著等特点。β-1,2-甘露聚糖是存在白念珠菌细胞表面的T-细胞依赖型多糖抗原，通常将它们与载体蛋白(BSA/TT)偶联形成糖蛋白疫苗，研究发现这类疫苗能诱导机体产生保护性抗体，但引起的免疫应答有限。我们拟将合成的β-1,2-甘露二糖、三糖和四糖通过磷酸乙醇胺基团(天然的糖蛋白连接方式)与强免疫原性载体蛋白钥孔虫戚雪蓝蛋白(KLH)偶联形成糖蛋白缀合物，以期得到高免疫原性的糖蛋白疫苗。
发明内容
本发明的第一个目的是针对现有技术中的不足，提供一种β-1,2-D-寡聚甘露糖蛋白缀合物。
本发明的第二个目的是，提供如上所述β-1,2-D-寡聚甘露糖蛋白缀合物的制备方法。
本发明的第三个目的是，提供如上所述β-1,2-D-寡聚甘露糖蛋白缀合物的用途。
本发明的第四个目的是，提供一种抗白念珠菌真菌疫苗。
为实现上述第一个目的，本发明采取的技术方案是：
一种β-1,2-D-寡聚甘露糖蛋白缀合物，所述β-1,2-D-寡聚甘露糖蛋白缀合物结构通式如下：
其中，n为0、1或2，R为载体蛋白KLH或HSA。
进一步，所述组成寡糖的单糖单元为β-1,2-D-甘露糖。
进一步，所述β-1,2-D-寡聚甘露糖蛋白缀合物由磷酸化的β-1,2-D-寡聚甘露糖与载体蛋白偶联制得。
进一步，所述β-1,2-D-寡聚甘露糖蛋白缀合物是以戊二酰基作为磷酸化的β-1,2-D-寡聚甘露糖和载体蛋白的连接基团。
为实现上述第二个目的，本发明采取的技术方案是：
如上所述β-1,2-D-寡聚甘露糖蛋白缀合物的制备方法，包括如下步骤：
第一步，合成β-1,2-D-甘露二糖受体7、β-1,2-D-甘露三糖受体10：
反应条件：a)TMSOTf，CH2Cl2，分子筛，-40℃，92.4％；b)CH3ONa，CH3OH，94.2％；c)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，79.5％(两步总收率)；d)TMSOTf，CH2Cl2，分子筛，-40℃，90.3％；e)CH3ONa，CH3OH，92.7％；f)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，77.6％(两步总收率)。
第二步，合成磷酸化β-1,2-D-寡聚甘露糖19a-c：
反应条件：a)TMSOTf，CH2Cl2，分子筛，-40℃，(12a，84.3％；12b，89.5％；12c，81.5％)；b)CH3ONa，CH3OH，91.3-95.5％；c)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，79.6-84.5％(两步总收率)；d)BnBr，NaH，TBAI，0℃，83.7-89.4％；e)TBAF，THF，83.4-88.2％；f)i)Tetrazole,CH2Cl2/CH3CN；ii)tert-BuOOH，79.3-87.5％(两步总收率)；g)DBU，CH2Cl2，86.3-93.2％。
第三步，合成β-1,2-D-寡聚甘露糖蛋白缀合物1a-c、2a-c：
反应条件:a)H2，Pd(OH)2/C，CH2Cl2/MeOH(1：1,v/v)，24小时，88.7-93.5％；b)DMF/PBS(4：1,v/v)，4小时；c)PBS，2.5天。
进一步，所述磷酸化β-1,2-D-寡聚甘露糖制备方法如下：
(1)以α-D-葡萄糖为原料，在反应助剂的作用下制备β-1,2-D-甘露单糖受体和3,4,6-三苄基-2-乙酰氧基葡萄糖三氯乙酰亚胺酯供体；
(2)上述制备的单糖供体和单糖受体在催化剂(TMSOTf)作用下发生糖基化反应制备二糖，在二糖的2’-位上通过构型翻转制备β-1,2-D-甘露二糖受体；β-1,2-D-甘露二糖受体和单糖供体通过前述方法制备β-1,2-D-甘露三糖受体；
(3)β-1,2-D-甘露糖受体分别和6-叔丁基二苯基硅基-3,4-二苄基-2-乙酰氧基葡萄糖三氯乙酰亚胺酯供体经糖基化作用制备相应β-1,2-D-寡聚甘露糖；寡聚甘露糖非还原端6-位脱掉TBDPS保护后和磷酸化试剂反应制得磷酸化β-1,2-D-寡聚甘露糖。
进一步，所述磷酸化β-1,2-D-寡聚甘露糖制备方法包括如下步骤：
(1)将磷酸化的β-1,2-D-寡聚甘露糖与羟琥珀酰亚胺戊二酸酯反应制得β-1,2-D-寡聚甘露糖活化酯；
(2)将步骤(1)制备的β-1,2-D-寡聚甘露糖活化酯在弱碱性条件下与载体蛋白偶联，得到β-1,2-D-寡聚甘露糖蛋白缀合物。
为实现上述第三个目的，本发明采取的技术方案是：
如上任一所述的β-1,2-D-寡聚甘露糖蛋白缀合物在制备预防和/或治疗真菌感染的药物中的应用。所述真菌为白念珠菌。
为实现上述第四个目的，本发明采取的技术方案是：
一种抗白念珠菌真菌疫苗，包括如上所述治疗有效量的β-1,2-D-寡聚甘露糖蛋白缀合物和药学上可接受的辅料或佐剂组成。
本发明优点在于：
1、将制备的化合物作为抗白念珠菌疫苗免疫小鼠，结果表明本发明的化合物能诱导机体产生较强的免疫应答，其中β-1,2-D-甘露三糖蛋白缀合物的免疫原性最强且产生的免疫血清能够特异性识别白念珠菌细胞表面抗原，从而可以起到预防白念珠菌感染的作用，本发明的β-1,2-D-寡聚甘露糖蛋白缀合物具有较强的应用价值。
附图说明
附图1是β-1,2-D-寡聚甘露糖蛋白缀合物通式。
附图2是β-1,2-D-甘露二糖、β-1,2-D-甘露三糖受体合成路线。反应条件：a)TMSOTf，CH2Cl2，分子筛，-40℃，92.4％；b)CH3ONa，CH3OH，94.2％；c)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，79.5％(两步总收率)；d)TMSOTf，CH2Cl2，分子筛，-40℃，90.3％；e)CH3ONa，CH3OH，92.7％；f)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，77.6％(两步总收率)。
附图3是磷酸化β-1,2-D-寡聚甘露糖二糖、三糖合成路线。反应条件：a)TMSOTf，CH2Cl2，分子筛，-40℃，(12a，84.3％；12b，89.5％；12c，81.5％)；b)CH3ONa，CH3OH，91.3-95.5％；c)i)DMSO，Ac2O；ii)L-selectride，THF，-78℃，79.6-84.5％(两步总收率)；d)BnBr，NaH，TBAI，0℃，83.7-89.4％；e)TBAF，THF，83.4-88.2％；f)i)Tetrazole,CH2Cl2/CH3CN；ii)tert-BuOOH，79.3-87.5％(两步总收率)；g)DBU，CH2Cl2，86.3-93.2％。
附图4是β-1,2-D-寡聚甘露糖二糖、三糖、四糖蛋白缀合物合成路线。反应条件:a)H2，Pd(OH)2/C，CH2Cl2/MeOH(1：1,v/v)，24小时，88.7-93.5％；b)DMF/PBS(4：1,v/v)，4小时；c)PBS，2.5天。
附图5是化合物2a的MALDI-TOF-MS谱图。
附图6是化合物2b的MALDI-TOF-MS谱图。
附图7是化合物2c的MALDI-TOF-MS谱图。
附图8是化合物1a-c免疫小鼠后检测血清中总抗体滴度，其中+Adj代表的是添加佐剂组别。
附图9是化合物1b第三次免疫小鼠后所得血清与白念珠菌细胞结合的免疫荧光图。
附图10是化合物1b第三次免疫小鼠后所得血清与白念珠菌细胞结合的流式细胞图。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明β-1,2-D-寡聚甘露糖蛋白缀合物(结构通式如图1所示)的制备反应路线(图2、图3、图4)：
第一步，合成β-1,2-D-甘露二糖(受体7)、β-1,2-D-甘露三糖受体(受体10)：
第二步，合成磷酸化β-1,2-D-寡聚甘露糖19a-c：
第三步，合成糖蛋白缀合物1a-c、2a-c：
实施例1化合物5的合成
将单糖受体3(1.23g,2.5mmol)(J.Org.Chem.2001,66,8411.)、供体4(1.95g,3.0mmol)(J.Carbohydr.Chem.1994,13,421.)以及活化的分子筛(1.0g)置于100mL圆底烧瓶并真空干燥半小时，然后将混合物用50mL无水二氯甲烷溶解。混悬液在氩气保护下于室温下搅拌半小时后冷却至-40℃，然后缓慢滴加三甲基硅基三氟甲磺酸酯(35.0μL,0.18mmol)。半小时后反应液用三乙胺中和淬灭并用硅藻土过滤，浓缩后所得初产品经硅胶柱层析(正己烷/乙酸乙酯,12:1,v/v)得白色泡沫状固体(2.23g,92.4％)。
1HNMR(400MHz,CDCl3)δ7.40-7.19(m,30H,Ar),5.88(m,1H,OCH2CH＝CH2),5.37(m,1H,OCH2CH＝CH 2),5.24-5.20(m,1H,OCH2CH＝CH 2),5.15(dd,J＝7.6Hz,9.6Hz,1H,H-2’),4.86-4.75(m,6H,H-1’,CH2Ph),4.56-4.43(m,7H,CH2Ph),4.42-4.39(m,1H,OCH 2CH＝CH2),4.34(s,1H,H-1),4.28(d,J＝3.2Hz,1H,H-2),4.22-4.01(m,1H,OCH 2CH＝CH2),3.82-3.74(m,3H,H-3,H-6,H-6’),3.73-3.57(m,5H,H-4,H-4’,H-5’,H-6’),3.52(d,J＝3.2Hz,H-3),3.44(m,1H),1.97(s,3H,Ac).ESI-MS:calcd.for C59H64O12[M+Na]+m/z,987.4；found,987.9.
实施例2化合物6的合成
将化合物5(1.85g,2.0mmol)用100mL无水甲醇溶解，加入甲醇钠(11.0mg,0.2mmol)并置于室温下搅拌过夜，反应完毕后加入离子交换树脂IR 120(H+form)中和，过滤除去树脂，浓缩后所得初产品经硅胶柱层析(正己烷/乙酸乙酯,4:1,v/v)得白色泡沫状固体(1.74g,94.2％)。
1H NMR(400MHz,CDCl3)δ7.55-7.23(m,30H,Ar),6.05-5.93(m,1H,OCH2CH＝CH2),5.40(m,1H,OCH2CH＝CH 2),5.29(m,1H,OCH2CH＝CH 2),5.17(dd,J＝11.2Hz,1H,OCH2Ph),5.05-4.93(m,4H,OCH2Ph),4.90(d,J＝11.2Hz,1H,OCH2Ph),4.81(d,J＝7.6Hz,1H,H-1’),4.72(d,J＝12.0Hz,1H,OCH2Ph),4.67-4.46(m,10H,OCH2Ph,OCH 2CH＝CH2,H-1),4.36(d,J＝3.2Hz,1H,H-2),4.17-4.10(m,1H,OCH 2CH＝CH2),4.01(t,J＝9.6Hz,1H,H-4),3.88-3.71(m,6H,H-2’,H-3’,H-6a’,H-6b’,H-6a,H-6b),3.70-3.57(m,3H,H-3,H-4’,H-5’),3.53-3.42(m,1H,H-5).13C NMR(100MHz,CDCl3)δ139.13,138.52,138.33,138.27,138.16,138.11,133.52,128.78,128.56,128.45,128.41,128.36,128.23,128.17,128.12,128.03,127.98,127.93,127.87,127.80,127.72,127.70,127.66,127.54,117.86,104.11,99.39,85.17,80.33,77.30,75.70,75.46,75.34,75.11,74.78,74.63,74.37,73.48,70.39,69.97,69.81,69.23.ESI-MS:calcd.for C57H62O11[M+K]+m/z,962.2；found,930.5.
实施例3化合物7的合成
将化合物6(1.38g,1.5mmol)置于100mL圆底烧瓶，然后加入二甲基亚砜(22.0mL)和醋酐(11.0mL)。反应液在室温下搅拌十八小时后用乙酸乙酯萃取,分别用饱和碳酸钠和食盐水洗涤，然后用无水硫酸钠干燥，过滤后浓缩得到的初产品用甲苯带旋两次。残留物用40mL无水四氢呋喃溶解并冷却至-78℃，氩气保护下缓慢滴加L-Selectride(1MTHF,7.5mL)并搅拌十五分钟，然后移除冷却装置并置于室温下继续搅拌十五分钟，甲醇淬灭反应后加入50mL二氯甲烷稀释，溶液分别用过氧化氢溶液(5％,30mL)、氢氧化钠溶液(1M,30mL)、硫代硫酸钠溶液(5％,30mL)和饱和食盐水(30mL)洗涤，经无水硫酸镁干燥、过滤及浓缩后的淡黄色油状液体，所得初产品经硅胶柱层析(正己烷/乙酸乙酯,4:1,v/v)得白色泡沫状固体(1.10g,79.5％)。
1H NMR(400MHz,CDCl3)δ7.55-7.23(m,30H,Ar),5.99-5.86(m,1H,OCH2CH＝CH2),5.27(m,1H,OCH2CH＝CH 2),5.21(m,1H,OCH2CH＝CH 2),5.03(s,1H,H-1’),5.02(dd,J＝10.8Hz,1H,OCH2Ph),4.99(dd,J＝11.2Hz,1H,OCH2Ph),4.94(dd,J＝10.8Hz,1H,OCH2Ph),4.89(dd,J＝10.8Hz,1H,OCH2Ph),4.69(dd,J＝10.8Hz,1H,OCH2Ph),4.68(dd,J＝11.6Hz,1H,OCH2Ph),4.64-4.59(m,2H,OCH2Ph),4.57(d,J＝3.2Hz,1H,H-2),4.56-4.48(m,4H,OCH2Ph),4.47(s,1H,H-1),4.46-4.43(m,1H,OCH 2CH＝CH2),4.41(d,J＝3.2Hz,1H,H-2’),4.11-4.06(m,1H,OCH 2CH＝CH2),3.98(t,J＝9.2Hz,1H,H-4’),3.91-3.70(m,5H,H-6a,H-4,H-6a’,H-6b,H-6b’),3.67-3.54(m,3H,H-3’,H-3,H-5’),3.51-3.44(m,1H,H-5).13C NMR(100MHz,CDCl3)δ138.44,138.31,138.18,133.83,128.38,128.27,128.16,128.01,127.95,127.78,127.72,127.65,117.18,100.15,99.35,81.42,80.34,75.6,75.14,74.44,74.07,73.50,73.39,70.85,70.75,70.07,69.40,67.70.ESI-MS:calcd.for C57H62O11[M+Li]+m/z,930.1；found,930.5.
实施例4化合物8的合成
将二糖受体7(0.95g,1.0mmol)、单糖供体4(0.78g,1.2mmol)以及活化的分子筛(0.25g)置于50mL圆底烧瓶并真空干燥半小时，然后将混合物用30mL无水二氯甲烷溶解。混悬液在氩气保护下于室温下搅拌半小时后冷却至-40℃，然后缓慢滴加三甲基硅基三氟甲磺酸酯(14.0μL,0.075mmol)。半小时后反应液用三乙胺中和淬灭并用硅藻土过滤，浓缩后所得初产品经硅胶柱层析(正己烷/乙酸乙酯,12:1,v/v)得白色泡沫状固体(1.26g,90.3％)。
1H NMR(600MHz,CDCl3)δ7.42-7.02(m,45H,Ar),5.86(m,1H,OCH2CH＝CH2),5.26(d,J＝8.0Hz,1H,H-1”),5.20-5.17(m,2H,H-2”,OCH2CH＝CH 2),5.11-5.07(m,1H,OCH2CH＝CH 2),4.98-4.94(m,3H,OCH2Ph),4.85-4.80(m,2H,OCH2Ph),4.75-4.69(m,4H,H-1’,OCH2Ph),4.63(d,J＝13.2Hz,1H,OCH2Ph),4.56-4.47(m,9H,H-2’,OCH2Ph),4.42(d,J＝12.0Hz,1H,OCH2Ph),4.41(s,1H,H-1),4.38-4.35(m,1H,OCH2CH＝CH2),4.20(d,J＝3.0Hz,1H,H-2),3.91(t,J＝8.4Hz,1H,H-3”),3.82-3.66(m,8H,H-4”,H-5’,H-5”,H-6a”,H-6a,H-6b,H-6a’,H-6b’),3.62(t,J＝9.6Hz,1H,H-4’),3.54-3.46(m,3H,H-3,H-3’,H-6a”),3.37(m,1H,H-5).MALDI-TOF-MS:calcd.for C86H92O17[M+Na]+m/z,1419.6,found 1420.0.
实施例5化合物9的合成
将化合物8(1.02g,0.75mmol)用50mL无水甲醇溶解，加入甲醇钠(11.0mg,0.2mmol)并置于室温下搅拌过夜，反应完毕后加入离子交换树脂IR 120(H+form)中和，过滤除去树脂，浓缩后所得初产品经硅胶柱层析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫状固体(0.94g,92.7％)。
1H NMR(400MHz,CDCl3)δ7.56-7.08(m,45H,Ar),6.01-5.89(m,1H,OCH2CH＝CH2),5.29(m,1H,OCH2CH＝CH 2),5.24(m,1H,OCH2CH＝CH 2),5.16-5.02(m,6H,OCH2Ph,H-1’),4.96(d,J＝10.8Hz,1H,OCH2Ph),4.88(d,J＝8.0Hz,1H,H-1”),4.81-4.66(m,3H,OCH2Ph),4.65-4.41(m,13H,OCH2Ph,H-2’,H-2,OCH 2CH＝CH2,H-1),4.15-4.07(m,1H,OCH 2CH＝CH2),4.01-3.71(m,9H,H-2”,H-3”,H-4’,H-6b”,H-6a”,H-6a,H-6b,H-6a’,H-6b’),3.68-3.56(m,5H,1H,H-5”,H-3’,H-4”,H-3,H-5),3.52-3.44(m,1H,H-5’).13C NMR(100MHz,CDCl3)δ139.21,138.68,138.59,138.44,138.39,138.16,138.11,133.79,128.50,128.41,128.36,128.31,128.25,128.20,128.11,128.06,127.92,127.86,127.83,127.73,127.69,127.64,127.59,127.48,127.43,127.27,117.43,105.35,100.08,99.93,86.73,80.25,80.03,77.18,75.52,75.44,75.31,75.25,74.99,74.83,74.72,74.62,74.09,73.66,73.46,73.33,71.16,70.31,70.19,69.77,69.45.MALDI-TOF-MS:calcd.for C84H90O16[M+Na]+m/z,1378.617；found,1378.115.
实施例6化合物10的合成
将化合物9(678.0mg,0.5mmol)置于50mL圆底烧瓶，然后加入二甲基亚砜(7.0mL)和醋酐(3.5mL)。反应液在室温下搅拌十八小时后用乙酸乙酯萃取,分别用饱和碳酸钠和食盐水洗涤，然后用无水硫酸钠干燥，过滤后浓缩得到的初产品用甲苯带旋两次。残留物用20mL无水四氢呋喃溶解并冷却至-78℃，氩气保护下缓慢滴加L-Selectride(1M THF,2.45mL)并搅拌十五分钟，然后移除冷却装置并置于室温下继续搅拌十五分钟，甲醇淬灭反应后加入20mL二氯甲烷稀释，溶液分别用过氧化氢溶液(5％,20mL)、氢氧化钠溶液(1M,20mL)、硫代硫酸钠溶液(5％,20mL)和饱和食盐水(20mL)洗涤，经无水硫酸镁干燥、过滤及浓缩后的淡黄色油状液体，所得初产品经硅胶柱层析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫状固体(526.3mg,77.6％)。
1H NMR(400MHz,CDCl3)δ7.66-6.94(m,45H,Ar),5.95-5.84(m,1H,OCH2CH＝CH2),5.25(m,1H,OCH2CH＝CH 2),5.20(s,1H,H-1”),5.19(m,1H,OCH2CH＝CH 2),5.17(s,1H,H-1’),5.06-4.96(m,4H,OCH2Ph),4.90(d,J＝10.8Hz,1H,OCH2Ph),4.75(br s,1H,H-2’),4.71-4.64(m,2H,OCH2Ph,H-2),4.61-4.42(m,10H,OCH2Ph,OCH 2CH＝CH2,H-1),4.38(d,J＝10.8Hz,1H,OCH2Ph),4.35(br s,1H,H-2”),4.28-4.21(m,1H,OCH2Ph),4.10-3.89(m,4H,H-4”,H-4’,OCH2Ph),3.88-3.49(m,13H,H-6a”,H-6b”,H-5”,H-3”,H-6a’,H-6b’,H-5’,H-3’,H-6a,H-6b,H-5,H-4,H-3),3.47-3.41(m,1H,H-5).13C NMR(100MHz,CDCl3)δ138.56,138.48,138.26,138.20,138.15,138.10,138.02,137.88,133.62,129.16,128.44,128.38,128.35,128.29,128.25,128.19,128.11,128.05,127.94,127.79,127.74,127.69,127.65,127.60,127.51,127.45,127.34,127.18,117.42,100.69,100.06,83.12,80.48,80.35,75.42,75.35,75.22,75.17,75.07,74.78,74.43,74.34,73.52,73.44,73.36,71.38,70.34,70.09,70.03,69.95,69.80,69.67,69.49,69.42,68.93,67.29.MALDI-TOF-MS:calcd.for C84H90O16[M+Na]+m/z,1378.617；found,1379.306.
实施例7化合物12a的合成
将单糖受体3(245mg,0.5mmol)、供体11(471.0mg,0.6mmol)(Eur.J.Org.Chem.1999,2523.)以及活化的分子筛(200mg)置于25mL圆底烧瓶并真空干燥半小时，然后将混合物用10mL无水二氯甲烷溶解。混悬液在氩气保护下于室温下搅拌半小时后冷却至-40℃，然后缓慢滴加三甲基硅基三氟甲磺酸酯(6.35μL,0.035mmol)。半小时后反应液用三乙胺中和淬灭并用硅藻土过滤，浓缩后所得初产品经硅胶柱层析(正己烷/乙酸乙酯,18:1,v/v)得白色泡沫状固体(478.5mg,84.3％)。
1H NMR(600MHz,CDCl3)δ7.71-7.01(m,35H,Ar),5.99-5.91(m,1H,OCH2CH＝CH2),5.36(m,1H,OCH2CH＝CH 2),5.21(m,1H,OCH2CH＝CH 2),5.16(dd,J＝9.0,8.0Hz,1H,H-2’),5.02(d,J＝8.0Hz,1H,H-1’),4.97-4.91(m,2H,OCH2Ph),4.81-4.73(m,3H,OCH2Ph),4.59(brs,2H),4.49-4.41(m,5H,OCH2Ph,OCH 2CH＝CH2,H-1),4.38(d,J＝3.6Hz,1H,H-2),4.08-4.01(m,2H,OCH 2CH＝CH2,H-6a’),3.65-3.60(m,1H,H-6b’),3.79(br d,J＝9.6Hz,1H,H-6a),3.75(t,J＝9.0Hz,1H,H-3’),3.64-3.59(m,2H,H-6b,H-4),3.56-3.49(m,4H,H-3,H-4’,H-5’),3.48-3.43(m,1H,H-5),1.95(s,3H),1.04(s,9H).13C NMR(150MHz,CDCl3)δ169.9,138.5,138.4,137.9,137.7,135.7,135.6,133.9,133.4,133.3,129.7,129.6,128.3,128.28,128.25,128.2,128.0,127.9,127.8,127.7,127.69,127.65,127.6,127.5,127.4,116.5,100.9,99.8,83.2,79.8,77.9,76.6,75.5,75.2,74.8,74.7,74.6,73.5,73.2,71.7,70.4,69.41,69.37,64.0,26.9,21.4,19.3.HRMS(ESI):calcd.for C68H76O12Si[M+Na]+m/z,1135.5004；found,1135.4979.
实施例8化合物12b的合成
由化合物7和化合物11制备，具体操作步骤同化合物12a的合成，产率89.5％。
1H NMR(600MHz,CDCl3)δ7.64-6.87(m,50H,Ar),5.87-5.78(m,1H,OCH2CH＝CH2),5.36(d,J＝8.4Hz,1H,H-1”),5.21(dd,J＝9.0,8.4Hz,1H,H-2”),5.20(br d,J＝10.8Hz,1H,OCH2CH＝CH 2),5.09(br d,J＝10.2Hz,1H,OCH2CH＝CH 2),4.98-4.89(m,3H,OCH2Ph),4.85(d,J＝12.0Hz,1H,OCH2Ph),4.77-4.72(m,2H,OCH2Ph,H-1’),4.68(q,J＝11.4Hz,2H,OCH2Ph),4.63(d,J＝12.8Hz,1H,OCH2Ph),4.59-4.44(m,8H,OCH2Ph,H-2’),4.43-4.36(m,3H,OCH2Ph,OCH 2CH＝CH2,H-1),4.23(d,J＝2.7Hz,1H,H-2),4.02-3.95(m,3H,OCH 2CH＝CH2,H-6a’,H-4),3.91(t,J＝9.0Hz,1H,H-3”),3.88-3.84(m,1H,H-6b”),3.79-3.58(m,7H,H-6a’,H-6b’,H-6a,H-6b,H-5’,H-4”,H-4’),3.54-3.45(m,3H,H-3’,H-3,H-5”),3.37-3.33(m,1H,H-5),1.96(s,3H),1.02(s,9H).13C NMR(150MHz,CDCl3)δ170.6,138.6,138.5,138.3,138.25,138.22,138.2,138.1,138.0,135.7,135.5,133.9,133.5,133.1,116.8,102.2,100.8,100.1,83.9,80.4,79.7,77.9,76.6,75.4,75.2,74.9,74.7,74.6,74.4,73.5,73.2,69.7,69.6,69.2,68.9,63.9,26.9,21.2,19.2.HRMS(ESI):calcd.forC95H104O17Si[M+Na]+m/z,1567.6942；found,1567.6924.
实施例9化合物12c的合成
由化合物10和化合物11制备，具体操作步骤同化合物12a的合成，产率81.5％。
1H NMR(600MHz,CDCl3)δ7.46-6.74(m,65H,Ar),5.79-5.73(m,1H,OCH2CH＝CH2),5.56(d,J＝7.8Hz,1H,H-1”’),5.23(t,J＝9.0,7.8Hz,1H,H-2”’),5.16(s,1H,H-1”),5.16-5.09(m,3H,OCH2CH＝CH 2,H-1”,H-1’),5.06(m,1H,OCH2CH＝CH 2),5.02(d,J＝11.4Hz,2H,OCH2Ph),4.95(d,J＝12.0Hz,1H,OCH2Ph),4.91(d,J＝10.8Hz,1H,OCH2Ph),4.85(d,J＝10.8Hz,1H,OCH2Ph),4.83(d,J＝3.0Hz,1H,H-2”),4.80-4.73(m,3H,OCH2Ph),4.66(q,J＝11.4Hz,2H,OCH2Ph),4.57-4.60(m,3H,OCH2Ph,H-2’),4.52-4.36(m,10H,OCH2Ph,H-2),4.34-4.30(m,1H,OCH 2CH＝CH2),4.27-4.24(m,2H,OCH2Ph,H-1),4.01-3.97(m,2H,H-4’,H-4”’),3.90-3.85(m,2H,OCH 2CH＝CH2,H-3”’),3.82-3.60(m,14H),3.56-3.54(m,1H,H-3’),3.44-3.38(m,2H,H-3,H-5’),3.31-3.28(m,1H,H-5),1.94(s,3H),0.94(s,9H).13C NMR(150MHz,CDCl3)δ170.6,138.67,138.6,138.5,138.4,138.3,138.2,137.9,137.8,137.3,135.64,135.62,133.5,133.1,132.8,129.4,129.3,128.4,128.3,128.3,128.22,128.20,128.17,128.11,128.1,127.9,127.88,127.86,127.8,127.7,127.6,127.5,127.49,127.44,127.40,127.36,127.33,127.2,126.9,117.2,101.8,100.7,100.5,100.4,84.0,81.90,80.6,77.4,75.8,75.5,75.3,75.2,75.1,75.00,74.9,74.8,74.7,74.5,74.4,73.65,73.4,73.3,73.1,72.7,71.2,70.6,70.2,69.6,69.4,69.3,69.0,68.1,62.8,26.9,21.2,19.2.HRMS(ESI):calcd.for C122H132O22Si[M+Na]+m/z,1999.8877；found,1999.8931.
实施例10化合物13a的合成
将化合物12a(470mg,4.22mmol)用15mL无水甲醇溶解，加入甲醇钠(4.5mg,0.84mmol)并置于室温下搅拌过夜，反应完毕后加入离子交换树脂IR 120(H+form)中和，过滤除去树脂，浓缩后所得初产品经硅胶柱层析(正己烷/丙酮,14:1,v/v)得白色泡沫状固体(417.5g,92.3％)。
1H NMR(600MHz,CDCl3)δ7.77-7.06(m,35H,Ar),6.02-5.95(m,1H,OCH2CH＝CH2),5.39(m,1H,OCH2CH＝CH 2),5.28(m,1H,OCH2CH＝CH 2),5.13(dd,J＝11.4Hz,1H,OCH2Ph),4.99(d,J＝10.8Hz,1H,OCH2Ph),4.95(d,J＝12.0Hz,1H,OCH2Ph),4.90(d,J＝12.0Hz,1H,OCH2Ph),4.88(d,J＝7.6Hz,1H,H-1’),4.85(d,J＝11.4Hz,1H,OCH2Ph),4.71(d,J＝12.0Hz,1H,OCH2Ph),4.62-4.50(m,6H,OCH2Ph,OCH 2CH＝CH2,H-1),4.43(d,J＝3.6Hz,1H,H-2),4.15-4.10(m,1H,OCH 2CH＝CH2),4.06(d,J＝10.8Hz,1H,H-6a’),4.02(t,J＝9.6Hz,1H,H-4),3.96-3.92(m,1H,H-6b’),3.86-3.80(m,3H,H-6a,H-6b,H-2’),3.76-3.71(m,1H,H-3’),3.65-3.52(m,3H,H-3,H-4’,H-5’),3.50-3.46(m,1H,H-5),1.09(s,9H).13C NMR(150MHz,CDCl3)δ139.2,138.5,138.25,138.21,137.8,135.7,135.6,133.57,133.51,133.5,129.64,129.60,128.4,128.34,128.29,128.1,128.0,127.99,127.8,127.73,127.70,127.6,127.57,127.5,117.8,103.7,99.6,85.2,80.3,76.9,75.7,75.2,74.9,74.8,74.3,73.9,73.5,70.1,70.0,69.1,63.9,26.9,19.3.HRMS(ESI):calcd.for C66H74O11Si[M+Na]+m/z,1093.4898；found,1093.4884.
实施例11化合物13b的合成
由化合物12b和甲醇钠制备，具体操作步骤同化合物13a的合成，产率95.5％。
1H NMR(600MHz,CDCl3)δ7.71-6.93(m,50H,Ar),5.92-5.86(m,1H,OCH2CH＝CH2),5.22(m,1H,OCH2CH＝CH 2),5.16(m,1H,OCH2CH＝CH 2),5.04(d,J＝11.4Hz,1H,OCH2Ph),4.95(d,J＝10.8Hz,1H,OCH2Ph),4.98-4.91(m,4H,OCH2Ph,H-1’),4.84(d,J＝7.8Hz,1H,H-1”),4.78(d,J＝10.8Hz,1H,OCH2Ph),4.63(d,J＝12.6Hz,1H,OCH2Ph),4.57(d,J＝11.4Hz,2H,OCH2Ph),4.54(d,J＝3.0Hz,1H,H-2’),4.51(d,J＝3.0Hz,1H,H-2),4.49-4.35(m,9H,OCH2Ph,OCH 2CH＝CH2,H-1),4.12-4.02(m,3H,OCH 2CH＝CH2,H-6a”,H-4),3.93-3.87(m,1H,H-6b”),3.83-3.72(m,5H,H-2”,H-4’,H-6a,H-6b,H-6a’),3.71-3.64(m,2H,H-6a’,H-3”),3.62-3.58(m,1H,H-5’),3.57-3.39(m,5H,H-3’,H-3,H-5’,H-4”,H-5),0.99(s,9H).13C NMR(150MHz,CDCl3)δ139.1,138.57,138.54,138.1,137.9,135.6,133.7,133.5,133.4,129.6,128.8,128.4,128.3,128.29,128.23,128.22,128.17,128.13,128.01,128.0,127.77,127.72,127.7,127.56,127.52,127.4,127.3,127.2,117.3,105.1,100.03,100.01,86.6,80.2,79.5,75.5,75.4,75.2,74.9,74.8,74.6,74.5,74.1,73.5,73.4,71.0,70.2,70.1,69.6,69.5,64.5,27.0,19.2.HRMS(ESI):calcd.for C93H102O16Si[M+Na]+m/z,1525.6835；found,1525.6777.
实施例12化合物13c的合成
由化合物12c和甲醇钠制备，具体操作步骤同化合物13a的合成，产率91.3％。
1H NMR(600MHz,CDCl3)δ7.64-6.86(m,65H,Ar),5.83-5.76(m,1H,OCH2CH＝CH2),5.31(s,1H,H-1”),5.15(m,1H,OCH2CH＝CH 2),5.09(m,1H,OCH2CH＝CH 2),5.06(d,J＝11.4Hz,1H,OCH2Ph),5.01(d,J＝10.8Hz,1H,OCH2Ph),4.98(s,1H,H-1’),4.97(d,J＝10.8Hz,1H,OCH2Ph),4.90(d,J＝11.4Hz,1H,OCH2Ph),4.85(d,J＝3.0Hz,1H,H-2’),4.84-4.78(m,3H,OCH2Ph),4.72(d,J＝7.8Hz,1H,H-1”’),4.62(d,J＝12.0Hz,1H,OCH2Ph),4.59(d,J＝11.4Hz,2H,OCH2Ph),4.52-4.39(m,10H,OCH2Ph,H-2’),4.37-4.32(m,3H,OCH2Ph,OCH 2CH＝CH2,H-1),4.22(d,J＝12.0Hz,1H,OCH2Ph),4.18(d,J＝10.8Hz,1H,OCH2Ph),4.02(t,J＝9.6Hz,1H,H-4”),3.95-3.88(m,4H,OCH 2CH＝CH2,H-4’,H-6a”’,H-6b”’),3.85-3.80(m,2H,H-6a”,H-6b”),3.75-3.55(m,10H),3.54-3.46(m,4H,H-3,H-2,H-5”’,H-5”),3.33-3.30(m,1H,H-5),0.96(s,9H).13C NMR(150MHz,CDCl3)δ139.1,138.8,138.6,138.5,138.34,138.30,138.2,138.1,138.0,135.7,135.6,133.7,133.5,133.3,129.5,128.4,128.4,128.3,128.26,128.24,128.2,128.14,128.09,128.0,127.97,127.93,127.8,127.77,127.73,127.6,127.5,127.49,127.4,127.36,127.3,127.2,127.0,117.2,105.7,101.4,100.3,99.7,86.2,81.0,80.3,79.8,76.6,75.8,75.7,75.6,75.5,75.4,75.34,75.32,75.2,74.8,74.7,74.5,73.4,72.2,70.2,70.1,70.0,69.99,69.91,69.7,69.4,69.2,63.9,26.9,19.3.HRMS(ESI):calcd.for C120H130O21Si[M+Na]+m/z,1957.8772；found,1957.8856.
实施例13化合物14a的合成
将化合物13a(380.0mg,0.36mmol)置于25mL圆底烧瓶，然后加入二甲基亚砜(5.0mL)和醋酐(2.5mL)。反应液在室温下搅拌十八小时后用乙酸乙酯萃取,分别用饱和碳酸钠和食盐水洗涤，然后用无水硫酸钠干燥，过滤后浓缩得到的初产品用甲苯带旋两次。残留物用20mL无水四氢呋喃溶解并冷却至-78℃，氩气保护下缓慢滴加L-Selectride(1MTHF,1.75mL)并搅拌十五分钟，然后移除冷却装置并置于室温下继续搅拌十五分钟，甲醇淬灭反应后加入20mL二氯甲烷稀释，溶液分别用过氧化氢溶液(5％,20mL)、氢氧化钠溶液(1M,20mL)、硫代硫酸钠溶液(5％,20mL)和饱和食盐水(20mL)洗涤，经无水硫酸镁干燥、过滤及浓缩后得淡黄色油状液体，初产品经硅胶柱层析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫状固体(323.5mg,84.5％)。
1H NMR(600MHz,CDCl3)δ7.69-7.07(m,35H,Ar),5.92-5.84(m,1H,OCH2CH＝CH2),5.26(m,1H,OCH2CH＝CH 2),5.16(m,1H,OCH2CH＝CH 2),5.12(br s,1H,H-1’),4.96-4.90(m,2H,OCH2Ph),4.88-4.82(m,2H,OCH2Ph),4.69(d,J＝3.6Hz,1H,H-2),4.67-4.62(m,2H,OCH2Ph)4.57(d,J＝12.0Hz,1H,OCH2Ph),4.51-4.37(m,5H,OCH2Ph,OCH 2CH＝CH2,H-1),4.36(d,J＝3.0Hz,1H,H-2’),4.09-4.03(m,2H,OCH 2CH＝CH2,H-6a’),3.91-3.79(m,4H,H-6b’,H-4,H-4’,H-6a),3.76-3.72(m,1H,H-6b),3.64-3.57(m,2H,H-3’,H-3),3.51-3.43(m,2H,H-5’,H-5),1.03(s,9H).13C NMR(150MHz,CDCl3)δ139.1,138.6,138.4,138.24,138.20,133.9,128.8,128.49,128.42,128.4,128.2,128.12,128.10,127.8,127.76,127.72,127.6,127.5,127.4,117.2,102.3,100.1,81.7,80.7,75.9,75.5,75.24,75.20,74.46,74.42,74.3,73.8,73.6,73.4,70.8,70.4,70.0,69.3,62.5.HRMS(ESI):calcd.forC66H74O11Si[M+Na]+m/z,1093.4898；found,1093.4882.
实施例14化合物14b的合成
由化合物13b经醋酐/二甲亚砜氧化、L-Selectride还原反应制备，具体操作步骤同化合物14a的合成，产率81.5％。
1H NMR(600MHz,CDCl3)δ7.65-6.89(m,50H,Ar),5.88-5.82(m,1H,OCH2CH＝CH2),5.29(s,1H,H-1”),5.20(m,1H,OCH2CH＝CH 2),5.16(m,1H,OCH2CH＝CH 2),5.12(s,1H,H-1’),4.94(d,J＝10.8Hz,2H,OCH2Ph),4.90(d,J＝12.0Hz,1H,OCH2Ph),4.85(d,J＝10.8Hz,1H,OCH2Ph),4.77(d,J＝10.8Hz,1H,OCH2Ph),4.73(d,J＝3.0Hz,1H,H-2’),4.63(d,J＝3.0Hz,1H,H-2),4.51-4.42(m,6H,OCH2Ph,H-1),4.41-4.37(m,3H,OCH2Ph,OCH 2CH＝CH2),4.34(d,J＝10.8Hz,1H,OCH2Ph),4.32(d,J＝2.4Hz,1H,H-2”),4.22(d,J＝10.8Hz,2H,OCH2Ph),4.06-3.97(m,3H,OCH2Ph,OCH 2CH＝CH2,H-6a”),3.92-3.86(m,2H,H-4”,H-4’),3.80-3.72(m,2H,H-6a’,H-6b’),3.69-3.55(m,5H,H-6a,H-6b,H-4,H-3’,H-3),3.54-3.47(m,3H,H-5”,H-5’,H-3”),3.41-3.37(m,1H,H-5).13C NMR(150MHz,CDCl3)δ138.5,138.4,138.2,138.1,138.1,138.07,138.03,137.9,135.63,135.61,133.6,133.5,133.4,129.5,128.9,128.5,128.3,128.3,128.2,128.2,128.1,128.0,127.9,127.85,127.82,127.7,127.69,127.65,127.63,127.5,127.4,127.4,127.2,127.1,117.3,100.6,100.3,99.6,83.1,80.4,79.9,76.6,75.4,75.3,75.1,74.8,74.8,74.5,74.1,73.4,70.8,70.3,70.2,70.1,69.9,69.6,69.3,69.0,67.5,64.1,26.9,19.3.HRMS(ESI):calcd.for C93H102O16Si[M+Na]+m/z,1525.6835；found,1525.6805.
实施例15化合物14c的合成
由化合物13c经醋酐/二甲亚砜氧化、L-Selectride还原反应制备，具体操作步骤同化合物14a的合成，产率79.6％。
1H NMR(600MHz,CDCl3)δ7.69-6.95(m,65H,Ar),5.85-5.78(m,1H,OCH2CH＝CH2),5.47(s,1H,H-1”’),5.26(s,1H,H-1”),5.19(m,1H,OCH2CH＝CH 2),5.11(m,1H,OCH2CH＝CH 2),5.01(s,1H,H-1’),4.98(d,J＝10.8Hz,1H,OCH2Ph),4.94-4.91(m,2H,OCH2Ph,H-2’),4.94-4.91(m,4H,OCH2Ph),4.68(d,J＝12.0Hz,1H,OCH2Ph),4.61-4.57(m,3H,OCH2Ph,H-2”’),4.50-4.39(m,7H,OCH2Ph),4.38(d,J＝3.0Hz,1H,H-2”),4.36-4.31(m,4H,OCH2Ph,H-2,H-1,OCH 2CH＝CH2),4.24-4.21(m,3H,OCH2Ph),4.09-4.01(m,5H,OCH2Ph,H-6a”’,H-6b”’,H-4”’,H-4”),3.95-3.92(m,1H,OCH 2CH＝CH2),3.62-3.60(m,10H),3.56-3.49(m,5H,H-3”,H-6a,H-5’,H-5”,H-3),3.35-3.32(m,1H,H-5),1.03(s,9H).13CNMR(150MHz,CDCl3)δ138.9,138.69,138.49,138.4,138.24,138.20,138.1,138.04,138.01,138.0,135.7,135.6,133.7,133.6,133.4,129.48,129.5,129.0,128.4,128.33,128.32,128.30,128.27,128.22,128.20,128.1,128.0,127.9,127.8,127.79,127.76,127.73,127.64,127.62,127.5,127.45,127.42,127.3,127.2,127.1,127.0,117.1,101.8,100.4,100.2,99.8,83.1,80.7,80.6,79.7,76.6,75.5,75.44,75.3,75.2,75.1,74.8,74.7,73.9,73.5,73.4,72.2,72.0,70.3,70.0,69.9,69.88,69.8,69.7,69.1,69.0,68.7,67.6,63.9,26.9,19.3.HRMS(ESI):calcd.for C120H130O21Si[M+Na]+m/z,1957.8772；found,1957.8750.
实施例16化合物15a的合成
将化合物14a(85.0mg,0.08mmol)溶于25mL N’N-二甲基甲酰胺并加入四丁基碘化铵(1.0mg,0.003mmol)和溴化苄(19μL,0.16mmol)，反应液冷却至0℃后加入氢化钠(4.8mg,0.12mmol)并在氩气保护下继续反应一小时，甲醇淬灭反应后加入50mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机层经无水硫酸镁干燥、过滤和浓缩后得初产品，初产品经硅胶柱层析(正己烷/丙酮,18:1,v/v)得白色泡沫状固体(77.0mg,83.7％)
1H NMR(600MHz,CDCl3)δ7.79-7.10(m,40H,Ar),6.02-5.93(m,1H,OCH2CH＝CH2),5.36(m,1H,OCH2CH＝CH 2),5.25(m,1H,OCH2CH＝CH 2),5.24(br s,1H,H-1’),5.21(d,J＝10.8Hz,1H,OCH2Ph),5.03-4.91(m,3H,OCH2Ph),4.79(d,J＝3.0Hz,1H,H-2),4.71-4.58(m,4H,OCH2Ph,H-1),4.57-4.53(m,1H,OCH 2CH＝CH2),4.52-4.44(m,1H,OCH2Ph),4.30(d,J＝3.0Hz,1H,H-2’),4.18-4.13(m,2H,OCH 2CH＝CH2,H-6a’),4.07-4.03(m,1H,OCH 2CH＝CH2,H-6b’),3.95-3.84(m,3H,H-4,H-4’,H-6a),3.80-3.76(m,1H,H-6b),3.73(d,J＝9.2,3.6Hz,1H,H-3),3.67(d,J＝9.2,3.2Hz,1H,H-3’),3.65-3.60(m,1H,H-5’),3.58-3.54(m,1H,H-5),1.12(s,9H).13C NMR(150MHz,CDCl3)δ139.5,138.53,138.51,138.5,138.4,135.7,135.6,133.9,133.7,133.5,129.6,129.58,128.7,128.6,128.37,128.35,128.3,128.2,128.04,128.02,127.9,127.72,127.70,127.6,127.5,127.4,127.1,117.1,100.3,100.8,82.3,80.5,77.4,75.6,75.2,75.1,75.0,74.2,74.1,73.7,73.5,70.7,70.6,70.2,69.6,69.5,64.4,26.9,19.3.HRMS(ESI):calcd.forC73H80O11Si[M+Na]+m/z,1183.5368；found,1183.5321.
实施例17化合物15b的合成
由化合物14b和溴化苄反应制备，具体操作步骤同化合物15a的合成，产率89.4％。
1H NMR(600MHz,CDCl3)δ7.69-6.93(m,55H,Ar),5.93-5.85(m,1H,OCH2CH＝CH2),5.41(s,1H,H-1”),5.24(m,1H,OCH2CH＝CH 2),5.21-5.15(m,3H,OCH2CH＝CH 2,OCH2Ph,H-1’),4.94(d,J＝11.4Hz,1H,OCH2Ph),4.96(d,J＝10.8Hz,1H,OCH2Ph),4.90-4.84(m,4H,OCH2Ph),4.81-4.75(m,2H,OCH2Ph,H-2’),4.65(d,J＝3.0Hz,1H,H-2),4.57(d,J＝11.4Hz,1H,OCH2Ph),4.52(d,J＝12.0Hz,1H,OCH2Ph),4.47-4.30(m,9H,H-1,OCH2Ph),4.20(br s,1H,H-2”),4.17-4.12(m,1H,OCH2Ph),4.08-3.98(m,4H,H-6a”,H-6b”,OCH 2CH＝CH2,OCH2Ph),3.91(t,J＝10.8,9.6Hz,1H,H-4”),3.84-3.76(m,2H,H-4’,H-6a),3.73-3.66(m,4H,H-4,H-6b,H-6a’,H-6b’),3.65-3.57(m,4H,H-3’,H-3,H-3”,H-5”),3.56-3.51(m,1H,H-5’),3.45-3.41(m,1H,H-5),1.01(s,9H).13C NMR(150MHz,CDCl3)δ140.0,138.8,138.6,138.53,138.50,138.3,138.2,138.1,137.9,135.7,133.8,133.7,133.6,129.4,128.6,128.4,128.3,128.24,128.20,128.17,128.15,128.1,127.99,127.95,127.8,127.7,127.66,127.60,127.56,127.54,127.51,127.46,127.3,127.2,127.0,126.8,117.2,101.5,100.7,83.4,80.6,80.2,75.37,75.34,75.2,75.1,74.9,74.8,74.8,74.6,74.4,74.3,73.4,73.3,70.8,70.5,70.2,70.18,69.9,69.7,69.1,68.9,64.4,26.9,19.3.HRMS(ESI):calcd.for C100H108O16Si[M+Na]+m/z,1615.7340；found,1615.7274.
实施例18化合物15c的合成
由化合物14c和溴化苄反应制备，具体操作步骤同化合物15a的合成，产率87.9％。
1H NMR(600MHz,CDCl3)δ7.69-6.97(m,70H,Ar),5.85-5.78(m,1H,OCH2CH＝CH2),5.49(s,1H,H-1”’),5.40(s,1H,H-1”),5.25(m,1H,OCH2CH＝CH 2),5.18(m,1H,OCH2CH＝CH 2),5.11(d,J＝10.8Hz,1H,OCH2Ph),5.03(s,1H,H-1’),4.99(d,J＝11.4Hz,1H,OCH2Ph),4.94-4.91(m,2H,OCH2Ph,H-2’),4.87-4.77(m,6H,OCH2Ph),4.65(d,J＝3.0Hz,1H,H-2”’),4.59(d,J＝12.0Hz,1H,OCH2Ph),4.54-4.34(m,13H,OCH2Ph,OCH 2CH＝CH2,H-2,H-1),4.32(d,J＝10.8Hz,1H,OCH2Ph),4.26(d,J＝3.0Hz,1H,H-2”),4.24(d,J＝10.8Hz,1H,OCH2Ph),4.13-4.17(m,2H,OCH2Ph),4.10-4.04(m,4H,OCH2Ph,H-6a”’,H-6b”’,H-4”),3.97-3.92(m,1H,OCH2Ph),3.86(t,J＝9.6Hz,1H,H-4’),3.80-3.59(m,11H),3.58-3.48(m,4H,H-6a,H-5”’,H-5”,H-3),3.37-3.33(m,1H,H-5),0.98(s,9H).13C NMR(150MHz,CDCl3)δ140.3,138.9,138.9,138.7,138.6,138.4,138.3,138.2,138.1,138.0,137.98,137.92,135.74,135.70,133.8,133.7,133.4,129.4,129.3,128.7,128.5,128.38,128.4,128.3,128.29,128.27,128.24,128.20,128.0,127.96,127.92,127.90,127.87,127.86,127.77,127.73,127.7,127.62,127.60,127.56,127.50,127.4,127.3,127.2,127.1,127.0,126.9,126.7,117.2,101.8,100.4,100.3,83.5,80.9,80.7,79.5,75.4,75.39,75.33,75.2,75.0,74.9,74.8,74.7,74.5,74.4,73.4,73.39,73.36,71.9,70.4,70.0,69.9,69.7,69.7,69.1,68.9,68.7,64.0,26.9,19.3.HRMS(ESI):calcd.for C127H136O21Si[M+Na]+m/z,2047.9241；found,2047.9266.
实施例19化合物16a的合成
将化合物15a(183.0mg,0.16mmol)溶于5mL四氢呋喃，然后加入四丁基氟化铵(0.63mL,1M)，室温下搅拌一天后加入30mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机层经无水硫酸镁干燥、过滤和浓缩后得初产品，初产品经硅胶柱层析(正己烷/丙酮,10:1,v/v)得白色泡沫状固体(77.0mg,83.4％)
1H NMR(600MHz,CDCl3)δ7.63-7.15(m,30H,Ar),5.97-5.88(m,1H,OCH2CH＝CH2),5.28(m,1H,OCH2CH＝CH 2),5.21(m,1H,OCH2CH＝CH 2),5.15(d,J＝12.0Hz,1H,OCH2Ph),5.04-4.96(m,5H,OCH2Ph,H-1’),4.93(d,J＝10.8Hz,1H,OCH2Ph),4.71(d,J＝10.8Hz,1H,OCH2Ph),4.66-4.60(m,2H,OCH2Ph),4.58-4.47(m,5H,OCH2Ph,H-1),4.43(d,J＝12.0Hz,1H,OCH2Ph),4.39(d,J＝3.0Hz,1H,H-2),4.29(d,J＝3.0Hz,1H,H-2),4.10-4.07(m,1H,OCH 2CH＝CH2),4.06(t,J＝9.6Hz,1H,H-4’),3.95(t,J＝9.6Hz,1H,H-4),3.92-3.84(m,2H,H-6a’,H-6b’),3.82(d,J＝10.8Hz,1H,H-6a),3.76(dd,J＝10.8,5.0Hz,1H,H-6b),3.66(dd,J＝9.3,2.9Hz,1H,H-3),3.59(dd,J＝9.6,3.0Hz,1H,H-3’),3.52-3.47(m,1H,H-5),3.44-3.41(m,1H,H-5’),13C NMR(150MHz,CDCl3)δ139.10,138.56,138.36,138.24,138.20,133.89,128.78,128.49,128.42,128.35,128.18,128.12,128.10,127.83,127.76,127.72,127.61,127.52,127.37,117.20,102.25,100.15,81.69,80.68,77.42,77.21,77.00,75.98,75.46,75.24,75.20,74.46,74.42,74.33,73.80,73.56,73.43,70.81,70.42,70.00,69.30,62.49.HRMS(ESI):calcd.forC57H62O11[M+Na]+m/z,945.4190；found,945.4161.
实施例20化合物16b的合成
由化合物15b和四丁基氟化铵反应制备，具体操作步骤同化合物16a的合成，产率88.2％。
1H NMR(600MHz,CDCl3)δ7.51-6.95(m,45H,Ar),5.92-5.84(m,1H,OCH2CH＝CH2),5.29(s,1H,H-1”),5.24(m,1H,OCH2CH＝CH 2),5.19-5.15(m,2H,OCH2CH＝CH 2,H-1’),5.07(d,J＝12.0Hz,1H,OCH2Ph),4.98(d,J＝11.4Hz,1H,OCH2Ph),4.93(d,J＝10.8Hz,1H,OCH2Ph),4.89-4.84(m,3H,OCH2Ph),4.71(d,J＝12.0Hz,1H,OCH2Ph),4.71(d,J＝3.0Hz,1H,H-2’),4.65(d,J＝12.0Hz,1H,OCH2Ph),4.61(d,J＝3.0Hz,1H,H-2),4.56(d,J＝10.8Hz,1H,OCH2Ph),4.46-4.40(m,6H,OCH2Ph,H-1,OCH 2CH＝CH2),4.29(d,J＝10.8Hz,1H,OCH2Ph),4.15(d,J＝3.0Hz,1H,H-2”),4.13(d,J＝11.4Hz,1H,OCH2Ph),4.07(d,J＝11.4Hz,1H,OCH2Ph),4.03-3.98(m,1H,OCH 2CH＝CH2),3.94(t,J＝9.6Hz,1H,H-4”),3.86(t,J＝9.6Hz,1H,H-4’),3.76-3.80(m,2H,H-6a”,H-6a’),3.75-3.67(m,5H,H-6b”,H-4,H-6a,H-6b,H-6b’),3.66-3.63(m,1H,H-3’),3.63-3.58(m,1H,H-3),3.55-3.51(m,2H,H-3”,H-5’),3.42-3.35(m,2H,H-5,H-5”).13C NMR(150MHz,CDCl3)δ139.6,138.7,138.5,138.4,138.4,138.2,138.1,138.0,137.7,133.6,128.6,128.41,128.40,128.4,128.3,128.29,128.23,128.2,128.04,128.00,127.8,127.7,127.69,127.65,127.61,127.6,127.4,127.1,127.1,117.4,101.6,100.7,100.4,83.0,80.7,80.6,75.5,75.4,75.2,75.1,74.7,74.66,74.62,74.6,74.5,73.6,73.4,71.6,70.5,70.4,69.9,69.8,69.6,68.7,62.4.HRMS(ESI):calcd.forC84H90O16[M+Na]+m/z,1377.6127；found,1377.6112.
实施例21化合物16c的合成
由化合物15c和四丁基氟化铵反应制备，具体操作步骤同化合物16a的合成，产率87.2％。
1H NMR(500MHz,CDCl3)δ7.52-6.02(m,60H,Ar),5.92-5.83(m,1H,OCH2CH＝CH2),5.53(s,1H,H-1”’),5.32(s,1H,H-1”),5.24(m,1H,OCH2CH＝CH 2),5.18(m,1H,OCH2CH＝CH 2),5.15(s,1H,H-1’),4.99(d,J＝12.0Hz,1H,OCH2Ph),5.00-4.88(m,5H,OCH2Ph,H-2’),4.83(d,J＝10.8Hz,1H,OCH2Ph),4.75(d,J＝12.0Hz,1H,OCH2Ph),4.87-4.77(m,15H,OCH2Ph,H-2,H-2”’,OCH 2CH＝CH2),4.25(d,J＝3.0Hz,1H,H-2”),4.18-4.10(m,2H,OCH2Ph),4.05-3.96(m,3H,OCH 2CH＝CH2,OCH2Ph),3.87-3.54(m,15H),3.48-3.40(m,2H,H-5,H-5”).13C NMR(125MHz,CDCl3)δ139.6,138.75,138.72,138.6,138.5,138.4,138.2,138.16,138.1,137.9,137.8,133.6,128.62,128.60,128.5,128.45,128.4,128.3,128.32,128.3,128.25,128.22,128.21,128.2,128.1,128.04,128.02,128.0,127.8,127.72,127.7,127.6,127.5,127.4,127.3,127.1,127.0,117.3,101.7,101.2,100.7,100.2,82.9,81.8,80.7,80.4,75.6,75.5,75.3,75.2,75.1,75.0,74.8,74.8,74.5,74.4,73.6,73.5,73.45,72.6,70.5,70.5,70.3,70.1,69.8,69.6,69.6,69.5,69.3,68.8,62.4.MALDI-TOF-MS:calcd.for C111H118O21[M+Na]+m/z,1811.10；found,1810.76.
实施例22化合物18a的合成
将化合物16a(50.0mg,0.06mmol)和磷酸化试剂17(138mg,0.26mmol)(Synthesis.1992,1269.)置于10mL圆底烧瓶并加入3.0mL二氯甲烷溶解，溶液冷却至0℃后滴加1H-四唑溶液(0.6mL,0.45M)，氩气保护下反应十五分钟后移去冷却装置继续在室温下搅拌两小时，然后冷却至-40℃后滴加过氧叔丁醇(0.07mL,5.5M)溶液，移除冷却装置继续室温下搅拌三小时后，加入15mL 10％硫代硫酸钠溶液淬灭反应，反应液用30mL二氯甲烷萃取，然后依次用饱和碳酸氢钠溶液(30mL)和食盐水(30mL)洗涤，有机层经无水硫酸镁干燥、过滤和浓缩后得初产品，初产品经硅胶柱层析(正己烷/乙酸乙酯,3:2,v/v)得非对映异构混合物(66.7mg,82.0％)。
Selected signals:1H NMR(500MHz,CDCl3)δ4.97(br s,1H),4.41(br s,1H).13CNMR(125MHz,CDCl3)δ102.14,102.09,100.31,100.26.31P NMR(160MHz,CDCl3)δ-0.61,-0.76.HRMS(ESI):calcd.for C81H84NO16P[M+Na]+m/z,1380.5425；found,1380.5381.
实施例23化合物18b的合成
由化合物16b和磷酸化试剂17反应制备，具体操作步骤同化合物18a的合成，产率87.5％。
Selected signals:1HNMR(500MHz,CDCl3)δ5.40(br s,1H),5.18(br s,1H),4.46(br s,1H).13C NMR(125MHz,CDCl3)δ101.62,101.52,100.79,100.70,100.65,100.54.31PNMR(160MHz,CDCl3)δ-0.50,-0.70.HRMS(ESI):calcd.for C108H112NO21P[M+Na]+m/z,1812.7362；found,1812.7319.
实施例24化合物18c的合成
由化合物16c和磷酸化试剂17反应制备，具体操作步骤同化合物18a的合成，产率79.3％。
NMR signals for 18c(isomer 1):1H NMR(600MHz,CDCl3)δ7.73-6.93(m,73H,Ar),5.85-5.78(m,1H,OCH2CH＝CH2),5.48(s,1H,H-1”’),5.30(s,1H,H-1”),5.19(m,1H,OCH2CH＝CH 2),5.14-5.10(m,1H,OCH2Ph),5.09(s,1H,H-1’),5.05-5.00(m,1H,OCH2Ph),4.97-4.82(m,9H,OCH2Ph,H-2’),4.71(d,J＝12.0Hz,1H,OCH2Ph),4.62(d,J＝12.0Hz,1H,OCH2Ph),4.59(d,J＝12.0 Hz,1H,OCH2Ph),4.56(d,J＝10.8 Hz,1H,OCH2Ph),4.57(d,J＝12.0 Hz,1H,OCH2Ph),4.53-4.44(m,6H,OCH2Ph,H-1,H-2”’),4.41-4.32(m,8H),4.29(d,J＝3.6 Hz,1H,H-2”),4.26(d,J＝10.8 Hz,1H,OCH2Ph),4.15-4.06(m,4H),4.03-3.93(m,4H),3.92-3.75(m,5H),3.72-3.58(m,8H),3.55(dd,J＝9.6,3.6 Hz,1H,OCH2Ph),3.52-3.43(m,2H),3.37-3.32(m,1H),3.26-3.18(m,1H),3.08-2.99(m,1H).13C NMR(150 MHz,CDCl3)δ156.18,144.08,143.92,141.16,141.12,139.49,138.64,138.52,138.43,138.34,138.21,138.05,138.02,137.99,137.84,137.75,135.80,136.76,133.59,128.64,128.57,128.52,128.45,128.32,128.28,128.26,128.22,128.18,128.16,128.12,128.04,128.02,127.97,127.96,127.88,127.85,127.81,127.70,127.64,127.56,127.54,127.50,127.43,127.39,127.24,127.11,127.06,126.97,126.95,125.21,125.12,119.77,117.31,102.08,101.31,100.55,100.29,83.10,81.97,80.53,80.35,75.52,75.31,75.27,75.12,75.05,74.69,74.58,74.24,74.20,73.61,73.56,73.41,73.21,70.92,70.54,70.19,70.18,69.69,69.60,69.51,69.36,69.15,69.12,68.77,66.89,66.85,66.57,66.45,47.04,40.95.31PNMR(160 MHz,CDCl3)δ-0.67.MALDI-TOF-MS:calcd.for C108H112NO21P[M+H]+m/z,2222.947；found,2223.236.NMR signals for 18c(isomer 2):1H NMR(600 MHz,CDCl3)δ7.73-6.93(m,73H,Ar),5.87-5.78(m,1H,OCH2CH＝CH2),5.48(s,1H,H-1”’),5.32(s,1H,H-1”),5.19(m,1H,OCH2CH＝CH 2),5.15-5.11(m,2H,OCH2Ph),5.07(s,1H,H-1’),5.06-5.00(m,3H,OCH2Ph),4.97-4.82(m,8H,OCH2Ph,H-2’),4.74(d,J＝11.4 Hz,1H,OCH2Ph),4.69(d,J＝12.0 Hz,1H,OCH2Ph),4.66(d,J＝12.0 Hz,1H,OCH2Ph),4.62(d,J＝10.8 Hz,1H,OCH2Ph),4.57(d,J＝12.0 Hz,1H,OCH2Ph),4.54(d,J＝12.0 Hz,1H,OCH2Ph),4.51(br s,1H,H-1),4.49(d,J＝10.8 Hz,2H,OCH2Ph),4.47-4.44(m,3H,OCH2Ph,H-2”’),4.41-4.34(m,8H),4.27(m,2H),4.26(d,J＝3.0 Hz,1H,H-2”),4.22(d,J＝10.8 Hz,2H,OCH2Ph),4.13-4.04(m,3H),4.03-3.93(m,3H),3.87-3.83(m,3H),3.68-3.58(m,9H),3.55(dd,J＝9.6,3.6 Hz,1H,OCH2Ph),3.52-3.47(m,2H),3.37-3.34(m,1H),3.14-3.08(m,2H).13CNMR(150 MHz,CDCl3)δ156.22,144.01,143.97,141.19,139.59,138.68,138.60,138.33,138.26,138.11,138.08,138.04,137.88,137.78,136.08,136.04,133.61,128.67,128.52,128.46,128.42,128.37,128.30,128.29,128.23,128.22,128.17,128.09,128.05,128.01,127.98,127.96,127.92,127.79,127.72,127.66,127.63,127.58,127.53,127.40,127.21,127.18,127.09,127.05,127.00,126.57,125.16,119.81,117.27,101.97,101.47,100.52,100.27,83.12,81.73,80.62,80.31,75.47,75.35,75.28,75.23,75.09,75.01,74.97,74.94,74.70,74.51,74.30,74.26,73.60,73.53,73.40,73.37,72.86,71.25,70.43,70.15,70.12,69.70,69.63,69.59,69.57,69.42,69.27,69.24,68.83,66.70,66.66,66.59,47.11,41.16,41.12.31P NMR(160MHz,CDCl3)δ-0.52.HRMS(ESI):calcd.for C108H112NO21P[M+Na]+m/z,2244.9293；found,2244.9475.
实施例25化合物19a的合成
将化合物18a(50.0mg,0.037mmol)置于5mL茄型瓶并加入0.8mL二氯甲烷溶解，室温下搅拌并滴加5滴1,8-二氮杂二环十一碳-7-烯(DBU)，两分钟后加入5滴乙酸中和反应，低温低压下除去溶剂后所得初产品经硅胶柱层析(二氯甲烷/甲醇,30:1,v/v)得非对映异构混合物(38.0mg,90.4％)。
Selected signals:1H NMR(600MHz,CDCl3)δ4.96(br s,1H),4.40(br s,1H).13CNMR(125MHz,CDCl3)δ101.84,101.69,100.14,99.86.HRMS(ESI):calcd.for C66H75NO14P[M+H]+m/z,1136.4920；found,1136.4925.
实施例26化合物19b的合成
由化合物18b和DBU反应制备，具体操作步骤同化合物19a的合成，产率93.2％。
Selected signals:1H NMR(600MHz,CDCl3)δ5.31(s,1H),5.13(s,1H),4.44(s,1H).13CNMR(150MHz,CDCl3)δ101.27,100.78,100.67,100.55,100.22,100,08.HRMS(ESI):calcd.for C93H102NO19P[M-H]-m/z,1566.6711；found,1566.6705.
实施例27化合物19c的合成
由化合物18c和DBU反应制备，具体操作步骤同化合物19a的合成，产率86.3％。
NMR signals for 19c(isomer 1):1H NMR(600MHz,CDCl3)δ7.50-6.92(m,65H,Ar),5.85-5.77(m,1H,OCH2CH＝CH2),5.46(s,1H,H-1”’),5.31(s,1H,H-1”),5.18(m,1H,OCH2CH＝CH 2),5.04(s,1H,H-1’),5.03-4.97(m,5H,OCH2Ph),4.90-4.78(m,5H,OCH2Ph),4.67-4.60(m,3H),4.59-4.45(m,6H,OCH2Ph),4.43-4.33(m,8H,OCH2Ph,H-1),4.31-4.19(m,3H),4.09(d,J＝11.2Hz,1H,OCH2Ph),4.04(d,J＝11.2Hz,1H,OCH2Ph),3.98-3.88(m,3H),3.84-3.68(m,6H),3.67-3.56(m,6H),3.54(dd,1H,J＝9.6,3.0Hz),3.51-3.45(m,1H),3.44-3.40(m,1H),3.37-3.32(m,1H).13C NMR(150MHz,CDCl3)δ138.78,138.49,138.24,138.16,138.11,138.05,137.99,137.87,137.71,133.59,128.76,128.56,128.50,128.42,128.40,128.31,128.30,128.24,128.17,128.16,128.11,128.07,128.04,128.00,127.98,127.90,127.85,127.76,127.73,127.69,127.64,127.51,127.40,127.20,127.15,127.10,117.25,101.65,101.59,100.40,100.10,83.33,81.50,80.66,80.26,75.46,75.36,75.27,75.23,75.03,74.99,74.95,74.73,74.59,74.35,73.58,73.51,73.38,70.25,70.08,69.78,69.52,69.48,69.40,69.03,68.83,66.60.NMRsignals for 19c(isomer 2):1H NMR(600MHz,CDCl3)δ7.55-6.93(m,65H,Ar),5.85-5.79(m,1H,OCH2CH＝CH2),5.53(s,1H,H-1”’),5.38(s,1H,H-1”),5.18(m,1H,OCH2CH＝CH 2),5.15-5.11(m,2H,OCH2Ph,H-1’),5.10(s,1H,H-1’),5.01-4.91(m,5H,OCH2Ph),4.89(d,J＝3.0Hz,1H),4.85-4.78(m,3H),4.66(d,J＝10.8Hz,1H,OCH2Ph),4.61-4.57(m,2H),4.47-4.37(m,14H,OCH2Ph,H-2,H-1),4.29(d,J＝10.8Hz,1H,OCH2Ph),4.25-4.14(m,3H),4.07(d,J＝11.2Hz,1H,OCH2Ph),3.98-3.75(m,7H),3.71-3.55(m,9H),3.51-3.44(m,2H),3.39-3.34(m,2H).13C NMR(150MHz,CDCl3)δ138.94,138.20,138.13,138.05,138.02,138.00,137.98,137.91,137.84,137.71,133.61,129.19,128.93,128.62,128.56,128.52,128.46,128.37,128.34,128.30,128.25,128.21,128.07,128.04,128.02,127.95,127.93,127.92,127.87,127.84,127.74,127.67,127.60,127.55,127.50,127.35,127.24,126.92,117.26,101.32,101.15,100.46,99.78,82.15,80.61,80.54,75.58,75.39,75.30,75.20,75.09,75.02,74.93,74.82,74.65,74.49,73.61,73.48,73.43,71.13,70.38,70.30,70.12,70.04,69.33,69.16,69.09,69.05,68.89,66.31,60.03.HRMS(ESI):calcd.for C120H131NO24P[M+H]+m/z,2000.8793；found,2000.8799.
实施例28化合物20a的合成
将化合物19a(20.0mg)和氢氧化钯碳(10Wt.％,10.0mg)置于5mL试管中并加入甲醇/二氯甲烷混合溶液(2mL,1:1,v:v)，然后将试管放入氢化反应釜中并在40个大气压下震荡反应一天，反应完毕后混悬液用硅藻土过滤，滤饼用甲醇洗涤，合并洗涤液并浓缩，残留物用乙酸乙酯洗涤后经LH-20柱层析得白色固体(8.2mg,91.5％)。
1H NMR(600MHz,CDCl3)δ4.84(s,1H,H-1’),4.58(s,1H,H-1),4.26-4.21(m,1H,),4.14-4.07(m,4H,H-2),4.05-4.02(m,1H,H-2’),3.90-3.84(m,2H,OCH 2CH2CH3),3.76-3.68(m,2H,H-4’),3.57-3.45(m,3H,OCH 2CH2CH3),3.42-3.38(m,1H,H-3’),3.33(s,1H,),3.30-3.27(m,3H,),3.24-3.12(m,4H,H-5’),1.64-1.57(m,2H,OCH2 CH 2CH3),0.94(t,J＝7.8Hz,1H,OCH2CH2 CH 3).13C NMR(150MHz,CDCl3)δ101.33,100.47,78.16,76.91,75.69,73.53,72.83,70.86,70.61,66.99,66.12,64.48,61.82,61.11,40.05,22.59,9.60.HRMS(ESI):calcd.forC17H33NO14P[M-H]-m/z,506.1639；found,506.1628
实施例29化合物20b的合成
由化合物19b经氢氧化钯碳催化还原反应制备，具体操作步骤同化合物20a的合成，产率88.7％。
1H NMR(600MHz,CDCl3)δ5.02(s,1H,H-1”),4.81(s,1H,H-1’),4.57(s,1H,H-1),4.31-4.25(m,1H,H-6”a),4.22(br s,1H,H-2’),4.20-4.15(m,1H,H-6”a),4.14-4.10(m,3H,H-2”,CH2OP),4.05(d,J＝3.0Hz,1H,H-2),3.89-3.84(m,3H,H-6a’,OCH 2CH2CH3),3.76-3.68(m,3H,H-6b’,H-4”,H-4’),3.55-3.42(m,6H),3.26-3.14(m,4H,H-5”,H-5,CH 2NH2),1.64-1.59(m,2H,OCH2 CH 2CH3),0.94(t,J＝7.8Hz,1H,OCH2CH2 CH 3).13C NMR(150MHz,CDCl3)δ101.4,101.3,100.3,79.4,77.7,76.86,75.4,75.3,73.3,72.8,70.8,70.4,67.7,66.4,66.1,64.7,64.6,61.9,61.1,60.8,40.0,22.6,9.6.HRMS(ESI):calcd.for C23H44NO19P[M-H]-m/z,668.2167；found,668.2166.
实施例30化合物20c的合成
由化合物19c经氢氧化钯碳催化还原反应制备，具体操作步骤同化合物20a的合成，产率93.5％。
1H NMR(600MHz,CDCl3)δ5.06(s,1H,H-1”’),5.01(s,1H,H-1”),4.75(s,1H,H-1’),4.57(s,1H,H-1),4.49(d,J＝3.0Hz,1H,H-2”),4.39-4.33(m,1H,H-6a”’),4.18-4.06(m,5H,H-6b”’,H-2’,H-2”’,CH2OP),4.05(d,J＝3.0Hz,1H,H-2”),3.89-3.81(m,5H),3.76-3.39(m,14H),3.37-3.29(m,2H,),3.24-3.11(m,5H,).13C NMR(150MHz,CDCl3)δ102.4,102.0,100.9,100.4,81.3,80.1,77.1,76.9,75.4,75.4,73.5,72.8,72.7,72.6,70.8,70.68,67.4,67.3,66.4,64.5,64.49,61.7,61.6,61.1,60.6,40.1,40.0,22.7,9.7.HRMS(ESI):calcd.for C29H54NO24P[M-H]-m/z,830.2695；found,830.2676.
实施例31化合物22a的合成
将化合物20a和琥珀酰亚胺戊二酸酯21(DSG,15equiv)溶于DMF/PBS(4:1,0.1MPBSbuffer)混合溶液，室温下搅拌四个小时，低温低压下除去溶剂并用甲苯带旋两次，残留物加入过量乙酸乙酯多次洗涤，干燥后得活化酯纯品，产率78.2％。
HRMS(ESI):calcd.for C26H42N2O19P[M-H]-m/z,717.2125,found,717.2119.
实施例32化合物22b的合成
由化合物20b和琥珀酰亚胺戊二酸酯21反应制备，具体操作步骤同化合物22a的合成，产率81.5％。
HRMS(ESI):calcd.for C32H52N2O24P[M-H]-m/z,879.2653,found,879.2656.
实施例33化合物22c的合成
由化合物20c和琥珀酰亚胺戊二酸酯21反应制备，具体操作步骤同化合物22a的合成，产率82.3％。
HRMS(ESI):calcd.for C38H62N2O29P[M-H]-m/z,1041.3187,found,1041.3176.
实施例34糖蛋白缀合物(1a-c,2a-c)的合成
将化合物22与载体蛋白KLH或HAS按照摩尔比为30:1(活化酯：载体蛋白)溶于0.1MPBS缓冲溶液，氩气保护下室温搅拌两天半。反应液经过Biogel A 0.5柱层析(0.1M PBS缓冲液,pH＝7.8)、去离子水透析及冷冻干燥后得纯品糖蛋白缀合物1a-c和2a-c(分子量见附图5-7)。
实施例35本发明化合物免疫活性测试
一、实验材料及来源
供试化合物：本发明实施例34中所制备的糖蛋白化合物1a-c。
二、试验方法
(一)小鼠免疫方案
1)选用6-8周雌性Balb/c小鼠(购于中国科学院上海实验动物中心)，按照疫苗+佐剂和仅用疫苗免疫方案分组，对照组为仅有佐剂组，每组6只小鼠；
2)将糖蛋白化合物1a-c及其相应添加佐剂组(溶于PBS)混悬液皮下注射小鼠，每次免疫的糖蛋白含有2μg的糖，每隔2周再用同样方法加强免疫，共免疫4次。分别于免疫前、第2次免疫后13天、第三次免疫后13天及第四次免疫后14天取血，分离血清并置于-80℃冰箱冻存待测。
(二)小鼠免疫前后血清中抗体滴度测定
各实验组每只小鼠混合血清滴度均采用ELISA方法检测。
1)包板：化合物2a-c分别用PH 9.6碳酸盐缓冲液稀释成5ug/ml包被于酶标排条板上。37℃结合2小时，后用封闭液(15％脱脂牛奶)200ul/孔4℃过夜；
2)PBST洗涤3次后，每孔加入100ul倍比稀释各组小鼠血清，37℃孵育2小时；
3)PBST洗涤5次后，加入100ul 1:2000稀释后的HRP标记的兔抗小鼠IgG二抗(Invitrogen)，37℃孵育45分钟；
4)PBST洗涤5次后，TMB显色后酶标仪(MultiscanMK3,Thermo)读取A450数值，高于阴性对照3倍的定义为阳性血清。
(三)小鼠免疫后所取血清与白念珠菌细胞的结合
1)挑取白念珠菌SC5314单克隆菌落用YPD液体培养基增菌24小时，此时为生长对数期的酵母样细胞。白念珠菌菌丝相诱导方法：将生长对数期白念珠菌利用PBS重悬洗涤2次，置于含有10％小牛血清1640培养基37℃静置培养90分钟；
2)将菌丝相或酵母相的白念珠菌用PBS重悬2次，用4％多聚甲醛-20℃固定10分钟，离心后再次用PBS重悬3次；
3)将实验组和对照组血清用含有10％小牛血清的PBS按1:100稀释后分别与固定后的菌体共孵育40分钟，其后用PBS离心、重悬3次；
4)用488标记的羊抗鼠IgG抗体与菌体共孵育30分钟，其后用PBS离心、重悬3次；
5)将重悬菌置于96孔板静置10分钟，利用荧光显微镜在488荧光下观察；
6)将重悬菌置于流式细胞离心管后直接上流式细胞仪检测，激发光选择FITC，利用阴性对照白念珠菌进行调零，再次将与荧光抗体共孵育后的菌进行上机检测，每次上样读取10000个细胞。
三、实验结果
本发明制备的KLH-糖蛋白缀合物1a-c皮下免疫小鼠，均能诱导机体产生较强的免疫应答，其中化合物1b的免疫原性最强且产生的免疫血清能够特异性识别白念珠菌细胞表面多糖抗原，实验结果见附图8-10。
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，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (10)

1.一种β-1,2-D-寡聚甘露糖蛋白缀合物，其特征在于，所述β-1,2-D-寡聚甘露糖蛋白缀合物结构通式为：
其中，n为0、1或2，R为载体蛋白KLH或HSA。
2.根据权要求1所述的β-1,2-D-寡聚甘露糖蛋白缀合物，其特征在于，组成寡糖的单糖单元为β-1,2-D-甘露糖。
3.根据权要求1所述的β-1,2-D-寡聚甘露糖蛋白缀合物，其特征在于，由磷酸化的β-1,2-D-寡聚甘露糖与载体蛋白偶联制得。
4.根据权要求3所述的β-1,2-D-寡聚甘露糖蛋白缀合物，其特征在于，以戊二酰基作为磷酸化的β-1,2-D-寡聚甘露糖和载体蛋白的连接基团。
5.权利要求1所述β-1,2-D-寡聚甘露糖蛋白缀合物的制备方法，其特征在于，包括如下步骤：
第一步，合成β-1,2-D-甘露二糖受体7、β-1,2-D-甘露三糖受体10：
第二步，合成磷酸化β-1,2-D-寡聚甘露糖19a-c：
第三步，合成β-1,2-D-寡聚甘露糖蛋白缀合物1a-c、2a-c：
6.根据权利要求5所述β-1,2-D-寡聚甘露糖蛋白缀合物的制备方法，其特征在于，所述磷酸化β-1,2-D-寡聚甘露糖制备方法如下：
(1)以α-D-葡萄糖为原料，在反应助剂的作用下制备β-1,2-D-甘露单糖受体和3,4,6-三苄基-2-乙酰氧基葡萄糖三氯乙酰亚胺酯供体；
(2)上述制备的单糖供体和单糖受体在催化剂(TMSOTf)作用下发生糖基化反应制备二糖，在二糖的2’-位上通过构型翻转制备β-1,2-D-甘露二糖受体；β-1,2-D-甘露二糖受体和单糖供体通过前述方法制备β-1,2-D-甘露三糖受体；
(3)β-1,2-D-甘露糖受体分别和6-叔丁基二苯基硅基-3,4-二苄基-2-乙酰氧基葡萄糖三氯乙酰亚胺酯供体经糖基化作用制备相应β-1,2-D-寡聚甘露糖；寡聚甘露糖非还原端6-位脱掉TBDPS保护后和磷酸化试剂反应制得磷酸化β-1,2-D-寡聚甘露糖。
7.根据权利要求5所述β-1,2-D-寡聚甘露糖蛋白缀合物的制备方法，其特征在于，包括如下步骤：
(1)将磷酸化的β-1,2-D-寡聚甘露糖与羟琥珀酰亚胺戊二酸酯反应制得β-1,2-D-寡聚甘露糖活化酯；
(2)将步骤(1)制备的β-1,2-D-寡聚甘露糖活化酯在弱碱性条件下与载体蛋白偶联，得到β-1,2-D-寡聚甘露糖蛋白缀合物。
8.权利要求1-4任一所述的β-1,2-D-寡聚甘露糖蛋白缀合物在制备预防和/或治疗真菌感染的药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述真菌为白念珠菌。
10.一种抗白念珠菌真菌疫苗，包括治疗有效量的权利要求1-4任一所述的β-1,2-D-寡聚甘露糖蛋白缀合物和药学上可接受的辅料或佐剂。
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