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β?1,2?D?寡聚甘露糖蛋白綴合物及其制備方法和應(yīng)用與流程

文檔序號:12242380閱讀:863來源:國知局
β?1,2?D?寡聚甘露糖蛋白綴合物及其制備方法和應(yīng)用與流程

本發(fā)明涉及抗真菌糖疫苗技術(shù)領(lǐng)域,具體涉及β-1,2-D-寡聚甘露糖蛋白綴合物及其制備方法,該類糖蛋白綴合物作為疫苗在白念珠菌感染的預(yù)防和治療中的應(yīng)用。



背景技術(shù):

近年來,白念珠菌感染引起的死亡率不斷上升,且病菌針對抗菌藥物產(chǎn)生嚴重的耐藥性,臨床上需要有效的預(yù)防和治療白念珠菌感染的藥物和策略。真菌感染的免疫治療主要采取的是主動性免疫治療,即利用抗原刺激機體產(chǎn)生強大的免疫應(yīng)答,通過產(chǎn)生抗體或分化效應(yīng)細胞達到清除感染的真菌或其它微生物的目的,具有毒副作用小、療效顯著等特點。β-1,2-甘露聚糖是存在白念珠菌細胞表面的T-細胞依賴型多糖抗原,通常將它們與載體蛋白(BSA/TT)偶聯(lián)形成糖蛋白疫苗,研究發(fā)現(xiàn)這類疫苗能誘導(dǎo)機體產(chǎn)生保護性抗體,但引起的免疫應(yīng)答有限。我們擬將合成的β-1,2-甘露二糖、三糖和四糖通過磷酸乙醇胺基團(天然的糖蛋白連接方式)與強免疫原性載體蛋白鑰孔蟲戚雪藍蛋白(KLH)偶聯(lián)形成糖蛋白綴合物,以期得到高免疫原性的糖蛋白疫苗。



技術(shù)實現(xiàn)要素:

本發(fā)明的第一個目的是針對現(xiàn)有技術(shù)中的不足,提供一種β-1,2-D-寡聚甘露糖蛋白綴合物。

本發(fā)明的第二個目的是,提供如上所述β-1,2-D-寡聚甘露糖蛋白綴合物的制備方法。

本發(fā)明的第三個目的是,提供如上所述β-1,2-D-寡聚甘露糖蛋白綴合物的用途。

本發(fā)明的第四個目的是,提供一種抗白念珠菌真菌疫苗。

為實現(xiàn)上述第一個目的,本發(fā)明采取的技術(shù)方案是:

一種β-1,2-D-寡聚甘露糖蛋白綴合物,所述β-1,2-D-寡聚甘露糖蛋白綴合物結(jié)構(gòu)通式如下:

其中,n為0、1或2,R為載體蛋白KLH或HSA。

進一步,所述組成寡糖的單糖單元為β-1,2-D-甘露糖。

進一步,所述β-1,2-D-寡聚甘露糖蛋白綴合物由磷酸化的β-1,2-D-寡聚甘露糖與載體蛋白偶聯(lián)制得。

進一步,所述β-1,2-D-寡聚甘露糖蛋白綴合物是以戊二?;鳛榱姿峄摩?1,2-D-寡聚甘露糖和載體蛋白的連接基團。

為實現(xiàn)上述第二個目的,本發(fā)明采取的技術(shù)方案是:

如上所述β-1,2-D-寡聚甘露糖蛋白綴合物的制備方法,包括如下步驟:

第一步,合成β-1,2-D-甘露二糖受體7、β-1,2-D-甘露三糖受體10:

反應(yīng)條件:a)TMSOTf,CH2Cl2,分子篩,-40℃,92.4%;b)CH3ONa,CH3OH,94.2%;c)i)DMSO,Ac2O;ii)L-selectride,THF,-78℃,79.5%(兩步總收率);d)TMSOTf,CH2Cl2,分子篩,-40℃,90.3%;e)CH3ONa,CH3OH,92.7%;f)i)DMSO,Ac2O;ii)L-selectride,THF,-78℃,77.6%(兩步總收率)。

第二步,合成磷酸化β-1,2-D-寡聚甘露糖19a-c:

反應(yīng)條件:a)TMSOTf,CH2Cl2,分子篩,-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%(兩步總收率);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%(兩步總收率);g)DBU,CH2Cl2,86.3-93.2%。

第三步,合成β-1,2-D-寡聚甘露糖蛋白綴合物1a-c、2a-c:

反應(yīng)條件:a)H2,Pd(OH)2/C,CH2Cl2/MeOH(1:1,v/v),24小時,88.7-93.5%;b)DMF/PBS(4:1,v/v),4小時;c)PBS,2.5天。

進一步,所述磷酸化β-1,2-D-寡聚甘露糖制備方法如下:

(1)以α-D-葡萄糖為原料,在反應(yīng)助劑的作用下制備β-1,2-D-甘露單糖受體和3,4,6-三芐基-2-乙酰氧基葡萄糖三氯乙酰亞胺酯供體;

(2)上述制備的單糖供體和單糖受體在催化劑(TMSOTf)作用下發(fā)生糖基化反應(yīng)制備二糖,在二糖的2’-位上通過構(gòu)型翻轉(zhuǎn)制備β-1,2-D-甘露二糖受體;β-1,2-D-甘露二糖受體和單糖供體通過前述方法制備β-1,2-D-甘露三糖受體;

(3)β-1,2-D-甘露糖受體分別和6-叔丁基二苯基硅基-3,4-二芐基-2-乙酰氧基葡萄糖三氯乙酰亞胺酯供體經(jīng)糖基化作用制備相應(yīng)β-1,2-D-寡聚甘露糖;寡聚甘露糖非還原端6-位脫掉TBDPS保護后和磷酸化試劑反應(yīng)制得磷酸化β-1,2-D-寡聚甘露糖。

進一步,所述磷酸化β-1,2-D-寡聚甘露糖制備方法包括如下步驟:

(1)將磷酸化的β-1,2-D-寡聚甘露糖與羥琥珀酰亞胺戊二酸酯反應(yīng)制得β-1,2-D-寡聚甘露糖活化酯;

(2)將步驟(1)制備的β-1,2-D-寡聚甘露糖活化酯在弱堿性條件下與載體蛋白偶聯(lián),得到β-1,2-D-寡聚甘露糖蛋白綴合物。

為實現(xiàn)上述第三個目的,本發(fā)明采取的技術(shù)方案是:

如上任一所述的β-1,2-D-寡聚甘露糖蛋白綴合物在制備預(yù)防和/或治療真菌感染的藥物中的應(yīng)用。所述真菌為白念珠菌。

為實現(xiàn)上述第四個目的,本發(fā)明采取的技術(shù)方案是:

一種抗白念珠菌真菌疫苗,包括如上所述治療有效量的β-1,2-D-寡聚甘露糖蛋白綴合物和藥學(xué)上可接受的輔料或佐劑組成。

本發(fā)明優(yōu)點在于:

1、將制備的化合物作為抗白念珠菌疫苗免疫小鼠,結(jié)果表明本發(fā)明的化合物能誘導(dǎo)機體產(chǎn)生較強的免疫應(yīng)答,其中β-1,2-D-甘露三糖蛋白綴合物的免疫原性最強且產(chǎn)生的免疫血清能夠特異性識別白念珠菌細胞表面抗原,從而可以起到預(yù)防白念珠菌感染的作用,本發(fā)明的β-1,2-D-寡聚甘露糖蛋白綴合物具有較強的應(yīng)用價值。

附圖說明

附圖1是β-1,2-D-寡聚甘露糖蛋白綴合物通式。

附圖2是β-1,2-D-甘露二糖、β-1,2-D-甘露三糖受體合成路線。反應(yīng)條件:a)TMSOTf,CH2Cl2,分子篩,-40℃,92.4%;b)CH3ONa,CH3OH,94.2%;c)i)DMSO,Ac2O;ii)L-selectride,THF,-78℃,79.5%(兩步總收率);d)TMSOTf,CH2Cl2,分子篩,-40℃,90.3%;e)CH3ONa,CH3OH,92.7%;f)i)DMSO,Ac2O;ii)L-selectride,THF,-78℃,77.6%(兩步總收率)。

附圖3是磷酸化β-1,2-D-寡聚甘露糖二糖、三糖合成路線。反應(yīng)條件:a)TMSOTf,CH2Cl2,分子篩,-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%(兩步總收率);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%(兩步總收率);g)DBU,CH2Cl2,86.3-93.2%。

附圖4是β-1,2-D-寡聚甘露糖二糖、三糖、四糖蛋白綴合物合成路線。反應(yīng)條件:a)H2,Pd(OH)2/C,CH2Cl2/MeOH(1:1,v/v),24小時,88.7-93.5%;b)DMF/PBS(4:1,v/v),4小時;c)PBS,2.5天。

附圖5是化合物2a的MALDI-TOF-MS譜圖。

附圖6是化合物2b的MALDI-TOF-MS譜圖。

附圖7是化合物2c的MALDI-TOF-MS譜圖。

附圖8是化合物1a-c免疫小鼠后檢測血清中總抗體滴度,其中+Adj代表的是添加佐劑組別。

附圖9是化合物1b第三次免疫小鼠后所得血清與白念珠菌細胞結(jié)合的免疫熒光圖。

附圖10是化合物1b第三次免疫小鼠后所得血清與白念珠菌細胞結(jié)合的流式細胞圖。

具體實施方式

下面結(jié)合具體實施方式,進一步闡述本發(fā)明。應(yīng)理解,這些實施例僅用于說明本發(fā)明而不用于限制本發(fā)明的范圍。此外應(yīng)理解,在閱讀了本發(fā)明記載的內(nèi)容之后,本領(lǐng)域技術(shù)人員可以對本發(fā)明作各種改動或修改,這些等價形式同樣落于本申請所附權(quán)利要求書所限定的范圍。

本發(fā)明β-1,2-D-寡聚甘露糖蛋白綴合物(結(jié)構(gòu)通式如圖1所示)的制備反應(yīng)路線(圖2、圖3、圖4):

第一步,合成β-1,2-D-甘露二糖(受體7)、β-1,2-D-甘露三糖受體(受體10):

第二步,合成磷酸化β-1,2-D-寡聚甘露糖19a-c:

第三步,合成糖蛋白綴合物1a-c、2a-c:

實施例1化合物5的合成

將單糖受體3(1.23g,2.5mmol)(J.Org.Chem.2001,66,8411.)、供體4(1.95g,3.0mmol)(J.Carbohydr.Chem.1994,13,421.)以及活化的分子篩(1.0g)置于100mL圓底燒瓶并真空干燥半小時,然后將混合物用50mL無水二氯甲烷溶解?;鞈乙涸跉鍤獗Wo下于室溫下攪拌半小時后冷卻至-40℃,然后緩慢滴加三甲基硅基三氟甲磺酸酯(35.0μL,0.18mmol)。半小時后反應(yīng)液用三乙胺中和淬滅并用硅藻土過濾,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,12:1,v/v)得白色泡沫狀固體(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=CH2),5.24-5.20(m,1H,OCH2CH=CH2),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,OCH2CH=CH2),4.34(s,1H,H-1),4.28(d,J=3.2Hz,1H,H-2),4.22-4.01(m,1H,OCH2CH=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.

實施例2化合物6的合成

將化合物5(1.85g,2.0mmol)用100mL無水甲醇溶解,加入甲醇鈉(11.0mg,0.2mmol)并置于室溫下攪拌過夜,反應(yīng)完畢后加入離子交換樹脂IR 120(H+form)中和,過濾除去樹脂,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,4:1,v/v)得白色泡沫狀固體(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=CH2),5.29(m,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.36(d,J=3.2Hz,1H,H-2),4.17-4.10(m,1H,OCH2CH=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.

實施例3化合物7的合成

將化合物6(1.38g,1.5mmol)置于100mL圓底燒瓶,然后加入二甲基亞砜(22.0mL)和醋酐(11.0mL)。反應(yīng)液在室溫下攪拌十八小時后用乙酸乙酯萃取,分別用飽和碳酸鈉和食鹽水洗滌,然后用無水硫酸鈉干燥,過濾后濃縮得到的初產(chǎn)品用甲苯帶旋兩次。殘留物用40mL無水四氫呋喃溶解并冷卻至-78℃,氬氣保護下緩慢滴加L-Selectride(1MTHF,7.5mL)并攪拌十五分鐘,然后移除冷卻裝置并置于室溫下繼續(xù)攪拌十五分鐘,甲醇淬滅反應(yīng)后加入50mL二氯甲烷稀釋,溶液分別用過氧化氫溶液(5%,30mL)、氫氧化鈉溶液(1M,30mL)、硫代硫酸鈉溶液(5%,30mL)和飽和食鹽水(30mL)洗滌,經(jīng)無水硫酸鎂干燥、過濾及濃縮后的淡黃色油狀液體,所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,4:1,v/v)得白色泡沫狀固體(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=CH2),5.21(m,1H,OCH2CH=CH2),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,OCH2CH=CH2),4.41(d,J=3.2Hz,1H,H-2’),4.11-4.06(m,1H,OCH2CH=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.

實施例4化合物8的合成

將二糖受體7(0.95g,1.0mmol)、單糖供體4(0.78g,1.2mmol)以及活化的分子篩(0.25g)置于50mL圓底燒瓶并真空干燥半小時,然后將混合物用30mL無水二氯甲烷溶解?;鞈乙涸跉鍤獗Wo下于室溫下攪拌半小時后冷卻至-40℃,然后緩慢滴加三甲基硅基三氟甲磺酸酯(14.0μL,0.075mmol)。半小時后反應(yīng)液用三乙胺中和淬滅并用硅藻土過濾,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,12:1,v/v)得白色泡沫狀固體(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=CH2),5.11-5.07(m,1H,OCH2CH=CH2),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.

實施例5化合物9的合成

將化合物8(1.02g,0.75mmol)用50mL無水甲醇溶解,加入甲醇鈉(11.0mg,0.2mmol)并置于室溫下攪拌過夜,反應(yīng)完畢后加入離子交換樹脂IR 120(H+form)中和,過濾除去樹脂,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫狀固體(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=CH2),5.24(m,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.15-4.07(m,1H,OCH2CH=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.

實施例6化合物10的合成

將化合物9(678.0mg,0.5mmol)置于50mL圓底燒瓶,然后加入二甲基亞砜(7.0mL)和醋酐(3.5mL)。反應(yīng)液在室溫下攪拌十八小時后用乙酸乙酯萃取,分別用飽和碳酸鈉和食鹽水洗滌,然后用無水硫酸鈉干燥,過濾后濃縮得到的初產(chǎn)品用甲苯帶旋兩次。殘留物用20mL無水四氫呋喃溶解并冷卻至-78℃,氬氣保護下緩慢滴加L-Selectride(1M THF,2.45mL)并攪拌十五分鐘,然后移除冷卻裝置并置于室溫下繼續(xù)攪拌十五分鐘,甲醇淬滅反應(yīng)后加入20mL二氯甲烷稀釋,溶液分別用過氧化氫溶液(5%,20mL)、氫氧化鈉溶液(1M,20mL)、硫代硫酸鈉溶液(5%,20mL)和飽和食鹽水(20mL)洗滌,經(jīng)無水硫酸鎂干燥、過濾及濃縮后的淡黃色油狀液體,所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫狀固體(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=CH2),5.20(s,1H,H-1”),5.19(m,1H,OCH2CH=CH2),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,OCH2CH=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.

實施例7化合物12a的合成

將單糖受體3(245mg,0.5mmol)、供體11(471.0mg,0.6mmol)(Eur.J.Org.Chem.1999,2523.)以及活化的分子篩(200mg)置于25mL圓底燒瓶并真空干燥半小時,然后將混合物用10mL無水二氯甲烷溶解?;鞈乙涸跉鍤獗Wo下于室溫下攪拌半小時后冷卻至-40℃,然后緩慢滴加三甲基硅基三氟甲磺酸酯(6.35μL,0.035mmol)。半小時后反應(yīng)液用三乙胺中和淬滅并用硅藻土過濾,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,18:1,v/v)得白色泡沫狀固體(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=CH2),5.21(m,1H,OCH2CH=CH2),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(br s,2H),4.49-4.41(m,5H,OCH2Ph,OCH2CH=CH2,H-1),4.38(d,J=3.6Hz,1H,H-2),4.08-4.01(m,2H,OCH2CH=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.

實施例8化合物12b的合成

由化合物7和化合物11制備,具體操作步驟同化合物12a的合成,產(chǎn)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=CH2),5.09(br d,J=10.2Hz,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.23(d,J=2.7Hz,1H,H-2),4.02-3.95(m,3H,OCH2CH=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.for C95H104O17Si[M+Na]+m/z,1567.6942;found,1567.6924.

實施例9化合物12c的合成

由化合物10和化合物11制備,具體操作步驟同化合物12a的合成,產(chǎn)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=CH2,H-1”,H-1’),5.06(m,1H,OCH2CH=CH2),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,OCH2CH=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,OCH2CH=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.

實施例10化合物13a的合成

將化合物12a(470mg,4.22mmol)用15mL無水甲醇溶解,加入甲醇鈉(4.5mg,0.84mmol)并置于室溫下攪拌過夜,反應(yīng)完畢后加入離子交換樹脂IR 120(H+form)中和,過濾除去樹脂,濃縮后所得初產(chǎn)品經(jīng)硅膠柱層析(正己烷/丙酮,14:1,v/v)得白色泡沫狀固體(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=CH2),5.28(m,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.43(d,J=3.6Hz,1H,H-2),4.15-4.10(m,1H,OCH2CH=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.

實施例11化合物13b的合成

由化合物12b和甲醇鈉制備,具體操作步驟同化合物13a的合成,產(chǎn)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=CH2),5.16(m,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.12-4.02(m,3H,OCH2CH=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.

實施例12化合物13c的合成

由化合物12c和甲醇鈉制備,具體操作步驟同化合物13a的合成,產(chǎn)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=CH2),5.09(m,1H,OCH2CH=CH2),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,OCH2CH=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,OCH2CH=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.

實施例13化合物14a的合成

將化合物13a(380.0mg,0.36mmol)置于25mL圓底燒瓶,然后加入二甲基亞砜(5.0mL)和醋酐(2.5mL)。反應(yīng)液在室溫下攪拌十八小時后用乙酸乙酯萃取,分別用飽和碳酸鈉和食鹽水洗滌,然后用無水硫酸鈉干燥,過濾后濃縮得到的初產(chǎn)品用甲苯帶旋兩次。殘留物用20mL無水四氫呋喃溶解并冷卻至-78℃,氬氣保護下緩慢滴加L-Selectride(1MTHF,1.75mL)并攪拌十五分鐘,然后移除冷卻裝置并置于室溫下繼續(xù)攪拌十五分鐘,甲醇淬滅反應(yīng)后加入20mL二氯甲烷稀釋,溶液分別用過氧化氫溶液(5%,20mL)、氫氧化鈉溶液(1M,20mL)、硫代硫酸鈉溶液(5%,20mL)和飽和食鹽水(20mL)洗滌,經(jīng)無水硫酸鎂干燥、過濾及濃縮后得淡黃色油狀液體,初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,6:1,v/v)得白色泡沫狀固體(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=CH2),5.16(m,1H,OCH2CH=CH2),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,OCH2CH=CH2,H-1),4.36(d,J=3.0Hz,1H,H-2’),4.09-4.03(m,2H,OCH2CH=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.for C66H74O11Si[M+Na]+m/z,1093.4898;found,1093.4882.

實施例14化合物14b的合成

由化合物13b經(jīng)醋酐/二甲亞砜氧化、L-Selectride還原反應(yīng)制備,具體操作步驟同化合物14a的合成,產(chǎn)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=CH2),5.16(m,1H,OCH2CH=CH2),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,OCH2CH=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,OCH2CH=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.

實施例15化合物14c的合成

由化合物13c經(jīng)醋酐/二甲亞砜氧化、L-Selectride還原反應(yīng)制備,具體操作步驟同化合物14a的合成,產(chǎn)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=CH2),5.11(m,1H,OCH2CH=CH2),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,OCH2CH=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,OCH2CH=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.

實施例16化合物15a的合成

將化合物14a(85.0mg,0.08mmol)溶于25mL N’N-二甲基甲酰胺并加入四丁基碘化銨(1.0mg,0.003mmol)和溴化芐(19μL,0.16mmol),反應(yīng)液冷卻至0℃后加入氫化鈉(4.8mg,0.12mmol)并在氬氣保護下繼續(xù)反應(yīng)一小時,甲醇淬滅反應(yīng)后加入50mL二氯甲烷稀釋,然后用30mL飽和食鹽水洗滌,有機層經(jīng)無水硫酸鎂干燥、過濾和濃縮后得初產(chǎn)品,初產(chǎn)品經(jīng)硅膠柱層析(正己烷/丙酮,18:1,v/v)得白色泡沫狀固體(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=CH2),5.25(m,1H,OCH2CH=CH2),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,OCH2CH=CH2),4.52-4.44(m,1H,OCH2Ph),4.30(d,J=3.0Hz,1H,H-2’),4.18-4.13(m,2H,OCH2CH=CH2,H-6a’),4.07-4.03(m,1H,OCH2CH=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.

實施例17化合物15b的合成

由化合物14b和溴化芐反應(yīng)制備,具體操作步驟同化合物15a的合成,產(chǎn)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=CH2),5.21-5.15(m,3H,OCH2CH=CH2,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”,OCH2CH=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.

實施例18化合物15c的合成

由化合物14c和溴化芐反應(yīng)制備,具體操作步驟同化合物15a的合成,產(chǎn)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=CH2),5.18(m,1H,OCH2CH=CH2),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,OCH2CH=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.

實施例19化合物16a的合成

將化合物15a(183.0mg,0.16mmol)溶于5mL四氫呋喃,然后加入四丁基氟化銨(0.63mL,1M),室溫下攪拌一天后加入30mL二氯甲烷稀釋,然后用30mL飽和食鹽水洗滌,有機層經(jīng)無水硫酸鎂干燥、過濾和濃縮后得初產(chǎn)品,初產(chǎn)品經(jīng)硅膠柱層析(正己烷/丙酮,10:1,v/v)得白色泡沫狀固體(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=CH2),5.21(m,1H,OCH2CH=CH2),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,OCH2CH=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.

實施例20化合物16b的合成

由化合物15b和四丁基氟化銨反應(yīng)制備,具體操作步驟同化合物16a的合成,產(chǎn)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=CH2),5.19-5.15(m,2H,OCH2CH=CH2,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,OCH2CH=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,OCH2CH=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.for C84H90O16[M+Na]+m/z,1377.6127;found,1377.6112.

實施例21化合物16c的合成

由化合物15c和四丁基氟化銨反應(yīng)制備,具體操作步驟同化合物16a的合成,產(chǎn)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=CH2),5.18(m,1H,OCH2CH=CH2),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”’,OCH2CH=CH2),4.25(d,J=3.0Hz,1H,H-2”),4.18-4.10(m,2H,OCH2Ph),4.05-3.96(m,3H,OCH2CH=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.

實施例22化合物18a的合成

將化合物16a(50.0mg,0.06mmol)和磷酸化試劑17(138mg,0.26mmol)(Synthesis.1992,1269.)置于10mL圓底燒瓶并加入3.0mL二氯甲烷溶解,溶液冷卻至0℃后滴加1H-四唑溶液(0.6mL,0.45M),氬氣保護下反應(yīng)十五分鐘后移去冷卻裝置繼續(xù)在室溫下攪拌兩小時,然后冷卻至-40℃后滴加過氧叔丁醇(0.07mL,5.5M)溶液,移除冷卻裝置繼續(xù)室溫下攪拌三小時后,加入15mL 10%硫代硫酸鈉溶液淬滅反應(yīng),反應(yīng)液用30mL二氯甲烷萃取,然后依次用飽和碳酸氫鈉溶液(30mL)和食鹽水(30mL)洗滌,有機層經(jīng)無水硫酸鎂干燥、過濾和濃縮后得初產(chǎn)品,初產(chǎn)品經(jīng)硅膠柱層析(正己烷/乙酸乙酯,3:2,v/v)得非對映異構(gòu)混合物(66.7mg,82.0%)。

Selected signals:1H NMR(500MHz,CDCl3)δ4.97(br s,1H),4.41(br s,1H).13C NMR(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.

實施例23化合物18b的合成

由化合物16b和磷酸化試劑17反應(yīng)制備,具體操作步驟同化合物18a的合成,產(chǎn)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)δ101.62,101.52,100.79,100.70,100.65,100.54.31P NMR(160MHz,CDCl3)δ-0.50,-0.70.HRMS(ESI):calcd.for C108H112NO21P[M+Na]+m/z,1812.7362;found,1812.7319.

實施例24化合物18c的合成

由化合物16c和磷酸化試劑17反應(yīng)制備,具體操作步驟同化合物18a的合成,產(chǎn)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=CH2),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)δ156.18,144.08,143.92,141.16,141.12,139.49,138.64,138.52,138.43,138.34,138.21,138.05,138.02,137.99,137.84,137.75,135.80,136.76,133.59,128.64,128.57,128.52,128.45,128.32,128.28,128.26,128.22,128.18,128.16,128.12,128.04,128.02,127.97,127.96,127.88,127.85,127.81,127.70,127.64,127.56,127.54,127.50,127.43,127.39,127.24,127.11,127.06,126.97,126.95,125.21,125.12,119.77,117.31,102.08,101.31,100.55,100.29,83.10,81.97,80.53,80.35,75.52,75.31,75.27,75.12,75.05,74.69,74.58,74.24,74.20,73.61,73.56,73.41,73.21,70.92,70.54,70.19,70.18,69.69,69.60,69.51,69.36,69.15,69.12,68.77,66.89,66.85,66.57,66.45,47.04,40.95.31P NMR(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=CH2),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.

實施例25化合物19a的合成

將化合物18a(50.0mg,0.037mmol)置于5mL茄型瓶并加入0.8mL二氯甲烷溶解,室溫下攪拌并滴加5滴1,8-二氮雜二環(huán)十一碳-7-烯(DBU),兩分鐘后加入5滴乙酸中和反應(yīng),低溫低壓下除去溶劑后所得初產(chǎn)品經(jīng)硅膠柱層析(二氯甲烷/甲醇,30:1,v/v)得非對映異構(gòu)混合物(38.0mg,90.4%)。

Selected signals:1H NMR(600MHz,CDCl3)δ4.96(br s,1H),4.40(br s,1H).13C NMR(125MHz,CDCl3)δ101.84,101.69,100.14,99.86.HRMS(ESI):calcd.for C66H75NO14P[M+H]+m/z,1136.4920;found,1136.4925.

實施例26化合物19b的合成

由化合物18b和DBU反應(yīng)制備,具體操作步驟同化合物19a的合成,產(chǎn)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.

實施例27化合物19c的合成

由化合物18c和DBU反應(yīng)制備,具體操作步驟同化合物19a的合成,產(chǎn)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=CH2),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=CH2),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.

實施例28化合物20a的合成

將化合物19a(20.0mg)和氫氧化鈀碳(10Wt.%,10.0mg)置于5mL試管中并加入甲醇/二氯甲烷混合溶液(2mL,1:1,v:v),然后將試管放入氫化反應(yīng)釜中并在40個大氣壓下震蕩反應(yīng)一天,反應(yīng)完畢后混懸液用硅藻土過濾,濾餅用甲醇洗滌,合并洗滌液并濃縮,殘留物用乙酸乙酯洗滌后經(jīng)LH-20柱層析得白色固體(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,OCH2CH2CH3),3.76-3.68(m,2H,H-4’),3.57-3.45(m,3H,OCH2CH2CH3),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,OCH2CH2CH3),0.94(t,J=7.8Hz,1H,OCH2CH2CH3).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

實施例29化合物20b的合成

由化合物19b經(jīng)氫氧化鈀碳催化還原反應(yīng)制備,具體操作步驟同化合物20a的合成,產(chǎn)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’,OCH2CH2CH3),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,CH2NH2),1.64-1.59(m,2H,OCH2CH2CH3),0.94(t,J=7.8Hz,1H,OCH2CH2CH3).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.

實施例30化合物20c的合成

由化合物19c經(jīng)氫氧化鈀碳催化還原反應(yīng)制備,具體操作步驟同化合物20a的合成,產(chǎn)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.

實施例31化合物22a的合成

將化合物20a和琥珀酰亞胺戊二酸酯21(DSG,15equiv)溶于DMF/PBS(4:1,0.1MPBSbuffer)混合溶液,室溫下攪拌四個小時,低溫低壓下除去溶劑并用甲苯帶旋兩次,殘留物加入過量乙酸乙酯多次洗滌,干燥后得活化酯純品,產(chǎn)率78.2%。

HRMS(ESI):calcd.for C26H42N2O19P[M-H]-m/z,717.2125,found,717.2119.

實施例32化合物22b的合成

由化合物20b和琥珀酰亞胺戊二酸酯21反應(yīng)制備,具體操作步驟同化合物22a的合成,產(chǎn)率81.5%。

HRMS(ESI):calcd.for C32H52N2O24P[M-H]-m/z,879.2653,found,879.2656.

實施例33化合物22c的合成

由化合物20c和琥珀酰亞胺戊二酸酯21反應(yīng)制備,具體操作步驟同化合物22a的合成,產(chǎn)率82.3%。

HRMS(ESI):calcd.for C38H62N2O29P[M-H]-m/z,1041.3187,found,1041.3176.

實施例34糖蛋白綴合物(1a-c,2a-c)的合成

將化合物22與載體蛋白KLH或HAS按照摩爾比為30:1(活化酯:載體蛋白)溶于0.1MPBS緩沖溶液,氬氣保護下室溫攪拌兩天半。反應(yīng)液經(jīng)過Biogel A 0.5柱層析(0.1M PBS緩沖液,pH=7.8)、去離子水透析及冷凍干燥后得純品糖蛋白綴合物1a-c和2a-c(分子量見附圖5-7)。

實施例35本發(fā)明化合物免疫活性測試

一、實驗材料及來源

供試化合物:本發(fā)明實施例34中所制備的糖蛋白化合物1a-c。

二、試驗方法

(一)小鼠免疫方案

1)選用6-8周雌性Balb/c小鼠(購于中國科學(xué)院上海實驗動物中心),按照疫苗+佐劑和僅用疫苗免疫方案分組,對照組為僅有佐劑組,每組6只小鼠;

2)將糖蛋白化合物1a-c及其相應(yīng)添加佐劑組(溶于PBS)混懸液皮下注射小鼠,每次免疫的糖蛋白含有2μg的糖,每隔2周再用同樣方法加強免疫,共免疫4次。分別于免疫前、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,分離血清并置于-80℃冰箱凍存待測。

(二)小鼠免疫前后血清中抗體滴度測定

各實驗組每只小鼠混合血清滴度均采用ELISA方法檢測。

1)包板:化合物2a-c分別用PH 9.6碳酸鹽緩沖液稀釋成5ug/ml包被于酶標排條板上。37℃結(jié)合2小時,后用封閉液(15%脫脂牛奶)200ul/孔4℃過夜;

2)PBST洗滌3次后,每孔加入100ul倍比稀釋各組小鼠血清,37℃孵育2小時;

3)PBST洗滌5次后,加入100ul 1:2000稀釋后的HRP標記的兔抗小鼠IgG二抗(Invitrogen),37℃孵育45分鐘;

4)PBST洗滌5次后,TMB顯色后酶標儀(MultiscanMK3,Thermo)讀取A450數(shù)值,高于陰性對照3倍的定義為陽性血清。

(三)小鼠免疫后所取血清與白念珠菌細胞的結(jié)合

1)挑取白念珠菌SC5314單克隆菌落用YPD液體培養(yǎng)基增菌24小時,此時為生長對數(shù)期的酵母樣細胞。白念珠菌菌絲相誘導(dǎo)方法:將生長對數(shù)期白念珠菌利用PBS重懸洗滌2次,置于含有10%小牛血清1640培養(yǎng)基37℃靜置培養(yǎng)90分鐘;

2)將菌絲相或酵母相的白念珠菌用PBS重懸2次,用4%多聚甲醛-20℃固定10分鐘,離心后再次用PBS重懸3次;

3)將實驗組和對照組血清用含有10%小牛血清的PBS按1:100稀釋后分別與固定后的菌體共孵育40分鐘,其后用PBS離心、重懸3次;

4)用488標記的羊抗鼠IgG抗體與菌體共孵育30分鐘,其后用PBS離心、重懸3次;

5)將重懸菌置于96孔板靜置10分鐘,利用熒光顯微鏡在488熒光下觀察;

6)將重懸菌置于流式細胞離心管后直接上流式細胞儀檢測,激發(fā)光選擇FITC,利用陰性對照白念珠菌進行調(diào)零,再次將與熒光抗體共孵育后的菌進行上機檢測,每次上樣讀取10000個細胞。

三、實驗結(jié)果

本發(fā)明制備的KLH-糖蛋白綴合物1a-c皮下免疫小鼠,均能誘導(dǎo)機體產(chǎn)生較強的免疫應(yīng)答,其中化合物1b的免疫原性最強且產(chǎn)生的免疫血清能夠特異性識別白念珠菌細胞表面多糖抗原,實驗結(jié)果見附圖8-10。

以上所述僅是本發(fā)明的優(yōu)選實施方式,應(yīng)當指出,對于本技術(shù)領(lǐng)域的普通技術(shù)人員,在不脫離本發(fā)明方法的前提下,還可以做出若干改進和補充,這些改進和補充也應(yīng)視為本發(fā)明的保護范圍。

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