幽门螺旋杆菌脂多糖外核心八糖的制备方法
技术领域
本发明涉及幽门螺旋杆菌脂多糖外核心八糖的制备方法,属于糖化学领域。
背景技术
幽门螺旋杆菌(Helicobacter Pylori,H.P.)在1994年被世界卫生组织列为I类致癌因子,它是一种革兰氏阴性菌,栖息在上皮细胞表面,被胃粘膜表面的粘液覆盖。世界上超过50%的人口上消化道感染了幽门螺旋杆菌,在发展中国家高达70%感染人群,在发达国家也达到20%~30%。急性感染中20%的病人发展为溃疡,1%的感染着甚至可能发展为胃癌或者粘膜相关淋巴瘤。2005年诺贝尔生理或医学奖授予澳大利亚两位科学家Warren和Mashall以表彰他们对于发现幽门螺旋杆菌在引发消化道疾病以及胃癌中的重要作用。目前最普遍的治疗幽门螺旋杆菌的方法是“三联疗法”,它主要利用质子泵抑制剂以及两种抗生素对幽门螺旋杆菌进行清除。但是用药后患者容易复发,且常常出现恶心、腹泻、假膜性肠炎等不良反应。伴随着抗生素的频繁使用,抗抗生素的幽门螺旋杆菌的菌株不断被发现,这也使得抗生素治疗幽门螺旋杆菌的效率逐渐降低。所以对于幽门螺旋杆菌疫苗的开发一直都是科学家研究的热点。
目前市场上还没有能防止幽门螺旋杆菌感染或者治疗幽门螺旋杆菌的疫苗存在。糖类疫苗作为疫苗开发的新的目标分子,越来越被科学家们重视,细菌表面的多糖结构往往对于细菌的致病性以及人体内免疫识别具有重要的作用,所以通过化学方法合成幽门螺旋杆菌表面寡多糖用于开发幽门螺旋杆菌疫苗具有非常重要的意义。
幽门螺旋杆菌细胞表面的寡糖为脂多糖(Lipopolysacchardie,LPS),它的LPS由O-抗原,核心结构,和脂质A三部分组成。幽门螺旋杆菌总共有6中血清型,O1-O6,它们的区别最主要是O-抗原糖部分的不同。不同血清型幽门螺旋杆菌的LPS的核心结构相对比较保守,所以通过化学合成其核心结构用作预防和治疗幽门螺旋杆菌感染的糖疫苗是非常好的一个选择。幽门螺旋杆菌核心结构包括内核心和外核心,外核心结构更容易暴露于环境,对于幽门螺旋杆菌感染引起体内的免疫反应有非常重要的作用。
目前研究的幽门螺旋杆菌脂多糖核心结构主要利用生物法从灭活的细菌中提取,其缺陷在于通常一次提取得到的产物量少,提取的分子结构也不均一,实验重复性差。所以我们选取幽门螺旋杆菌外核心八糖为我们的目标分子进行化学全合成,通过化学合成,我们可以大量的得到分子均一、结构确定的目标结构。
发明内容
本发明所要解决的技术问题是通过化学方法合成幽门螺旋杆菌外核心八糖,这个八糖结构中含有五个葡萄糖1,2-α糖苷键的合成是目标八糖的关键步骤。本发明利用远程邻基参与效应、溶剂效应、温度效应,添加剂和离去基团的协同作用,提高糖基化过程的α选择性。此协同作用成功的解决了葡萄糖α糖苷键的形成的难题。利用合成的葡萄糖糖砌块,半乳糖糖砌块,庚糖糖砌块,合成保护的最后的目标八糖,最后脱去保护得到目标八糖如式1。同时,八糖的还原端为带有氨基的连接臂,为以后连接蛋白制成糖缀合物疫苗做了准备。
其中,R为-(CH2)n-N-Y1Y2或者-(CH2)n-N-Y1Y2(linker),n=1~10,Y1为H或者为卞基(Bn),Y2为H或者卞甲氧羰基(Cbz)。R1,R2,R3,R6,R7,R8,R10,R12,R14,R15,R16,R18,R20,R21,R23,R24,R26为H或者苄基(Bn);R5,R11,R17,R19,R22,R25为H或者2-萘甲基(Nap);R4,R9,R13,R27为酯基。N为氮。
本发明还提供式Ⅰ所示化合物的合成方法,具体包括以下步骤:
步骤一,合成葡萄糖糖砌块A,糖砌块A的结构如式Ⅱ所示,其端基位为N-苯基-三氟乙酰亚胺酯,为离去基团;其余取代基Rn参见式Ⅰ。
具体地,单糖砌块A的合成方法包括:以全乙酰化葡萄糖为起始原料,在路易斯酸的作用下与对甲苯硫酚作用生成全乙酰硫糖苷化合物,在碱性条件下脱去乙酰基,裸露四个羟基,所得四羟基化合物与苯甲醛二甲缩醛(PhCH(OCH3)2)在对甲苯磺酸(p-TsOHH2O)的催化下生成4,6-苄叉基保护的硫糖苷化合物;把4,6-苄叉基保护的硫糖苷化合物的3-OH用2-甲基萘(2-Nap)保护,2-OH用苄基(Bn)保护后得到全
保护化合物,然后在硼烷(BH3THF)和路易斯酸的作用下选择性打开4,6-卞叉基,裸露6-OH,裸露的6-OH再用酰基保护,得到全保护的葡萄糖硫糖苷化合物,全保护的葡萄糖硫糖苷化合物在NBS或者NIS条件下水解,裸露端基位OH,端基位-OH与CF3CNPhCl反应,得到葡萄糖砌块A。
步骤二,合成半乳糖糖砌块B,糖砌块B的结构如式Ⅲ所示,其端基位为叔丁基二丁基硅烷基(TBS)保护,其余取代基Rn参见式Ⅰ。
具体地,单糖砌块B的合成方法包括:以化合物2,3-二-丙叉-6-卞基乙硫半乳糖为起始原料,2-OH用Bz保护,得到2-OBz化合物,2-OBz化合物在酸作用下打开丙叉基,得到2,3-二羟基化合物,2,3-二羟基化合物的3-OH选择性的用Bn保护得到3-OBn化合物,3-OBn化合物的4-OH用Lev保护,得到4-Lev硫糖苷化合物,4-Lev硫糖苷化合物在NBS条件下水解,裸露端基位OH,端基位-OH与叔丁基二甲基氯硅烷(TBSCl)反应,得到端基TBS全保护的半乳糖,在醋酸肼作用下脱去Lev得到半乳糖砌块B。
步骤三,合成庚糖砌块C、D,如式Ⅳ所示,糖砌块C端基位为乙硫基保护,R17为Nap保护;糖砌块D端基位为linker,例如O-(CH2)n-N(Bn)Cbz,或者O-(CH2)n-SBn,或者O-(CH2)n-N3;R17为酰基保护;其余取代基Rn参见式I。
具体地,单糖砌块C,D的合成方法包括:以2,3-O-丙叉基-4-O-卞基甘露乙硫糖苷为起始原料,经过swern氧化,得到相应的醛,然后经过witting烯化延长6位的碳链,得到6位脱氧的烯烃化合物,烯烃化合物经过双羟基化得到6,7-二-羟基化合物,在7-OH上利用二丁基氧化锡(Bu2SnO)选择性的用Nap保护得到7-Nap化合物,6-OH用Bn保护得到全保护化合物;将全保护化合物在80%的醋酸作用下脱去丙叉基后得到2,3-二-羟基化合物,利用二丁基氧化锡(Bu2SnO)和Nap选择性的保护3-OH,得到庚糖砌块C;糖砌块C的2-OH用Lev保护后得到2-Lev化合物,2-Lev化合物与连接臂(linker)反
应,得到端基位连接臂的庚糖化合物,7-Nap在DDQ的作用下离去得到7-OH化合物,7-OH化合物用乙酰基保护得到化合物7-OAc,最后在醋酸肼的作用下脱去Lev得到庚糖砌块D。
步骤四,组装三糖供体:利用糖砌块合成三糖砌块G(式Ⅴ),糖基化反应的条件反应溶剂为Et
2O/DCM,加入相当于供体10当量的噻酚,使用4
分子筛为干燥剂,反应温度为0℃,反应时间为3~5h,反应过程都为氩气保护。反应用TLC板监控,当反应结束后用吡啶(Pyridine)淬灭反应,产物用硅胶柱纯化。三糖G端基位为N-苯基-三氟乙酰亚胺酯为离去基团,在八糖组装中作为糖基供体。
步骤五,组装五糖受体:利用糖砌块合成五糖砌块H(式Ⅵ),氟糖苷糖基化反应的条件:溶剂为Et
2O/DCM,加入相当于供体10当量的噻酚,使用4
分子筛为干燥剂,反应温度为0℃,反应时间为3~5h,反应过程都为氩气保护。反应用TLC板监控,当反应结束后用吡啶淬灭反应,产物用硅胶柱纯化。硫糖苷在糖基化反应过程使用NIS/TMSOTf或者NIS/TfOH作为促进剂,DCM作为溶剂,4
分子筛作为干燥剂。合成的五糖在八糖组装中作为糖基受体。
步骤六,组装八糖受体:八糖的组装采取[3+5]合成策略。反应溶剂为DCM,促进剂为TMSOTf,反应温度为0℃,反应时间为7h,反应过程都为氩气保护。反应用TLC板监控,当反应结束后用吡啶(Pyridine)淬灭反应,产物用硅胶柱纯化。
步骤七,八糖脱保护:保护八糖在碱性条件下脱去酰基,经过硅胶柱纯化后,使用Pd/C,H2脱去反应3天脱去芳香基团,待芳香基团全托保护后,用反相C18柱纯化,最后得到目标八糖分,如式Ⅷ。
本发明还提供一种生成葡萄糖α糖苷键的方法,糖基供体和受体在甲苯中共蒸2~3次,加入干燥的DCM或者DCM/Et
2O;底物浓度为0.01-0.1M,以活化的3
或者4
分子筛为干燥剂,加入相当于糖基供体8~10当量的噻酚;在室温下搅拌20~30min后冷却至-50~0℃,加入TMSOTf为促进剂,反应时间为1~7h;用吡啶终止反应。
具体地,糖基供体和受体在甲苯中共蒸3次,加入干燥的DCM或者体积比为1:2的DCM/Et
2O;底物浓度为0.02-0.05M,以活化的3
或者4
分子筛为干燥剂,加入相当于糖基供体10当量的噻酚;在室温下搅拌20~30min后冷却至0℃,加入TMSOTf为促进剂,反应时间为3-5h;用吡啶终止反应。
本发明有益效果:
本发明是通过化学合成得幽门螺旋杆菌外核心八糖。本发明中通过保护基策略,溶剂效应,温度效应,发现了一种非常有利于葡萄糖α糖苷键形成的方法,并将此方法应用到幽门螺旋杆菌外核心八糖的合成中。合成的幽门螺旋杆菌外核心八糖与抗原蛋白相连,制成糖缀合物,对于发展预防和治疗幽门螺旋杆菌疫苗具有重要的作用。
附图说明
图1:糖砌块8的合成。
图2:糖砌块21的合成。
图3:糖砌块28的合成。
图4:三糖供体的32组装。
图5:五糖受体的42组装。
图6:目标八糖41的合成。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
本发明的产率的计算方法为“产物(mol)/反应底物(mol)*100%。本发明中化合物结构鉴定的方法为核磁共振图谱的测定(400M/600M/700M),高分辨质谱测定,旋光值测定,结果已在每个化合物的具体合成中列出。
实施例1
糖砌块8的合成如图1:
如图1所示,以全乙酰化葡萄糖1为起始原料,在三氟化硼乙醚(BF3OEt2)的作用下与对甲苯硫酚作用生成全乙酰硫糖苷化合物2。乙酰基在甲醇钠(NaOCH3)作用下脱去乙酰基,裸露四个羟基。四羟基化合物与苯甲醛二甲缩醛(PhCH(OCH3)2)在对甲苯磺酸(p-TsOH H2O)的催化下生成4,6-苄叉基保护的硫糖苷3化合物。选择性的把3-OH用2-甲基萘(2-Nap)保护得到化合物4,2-OH用苄基(Bn)保护后得到化合物5。利用硼烷(BH3THF)和三氟甲磺酸三甲基硅脂(TMSOTf)作用下选择性打开4,6-卞叉基,裸露6-OH得到化合物6。化合物6的6-OH用苯甲酰基(Bz)保护,得到全保护的葡萄糖硫糖苷化合物7。硫糖苷在NBS条件下水解,裸露端基位OH,端基位-OH与CF3CNPhCl反应,得到葡萄糖砌块8。
具体试验操作和步骤:
化合物2:全乙酰葡萄糖1(10.0g,25.6mmol)溶解在DCM(128mL)。在0℃下加入对甲苯硫酚(4.8g,38,6mmol)和BF3·OEt2(4.8mL,38.4mmol)。反应液恢复到室温,搅拌12个小时。反应液用DCM稀释后用饱和NaHCO3洗涤,有机相拥无水Na2SO4干燥,浓缩后用柱层析分离纯化(石油醚/乙酸乙酯,3:1),得到化合物2(10.1g,87%)。[α]22
D=11.6(c1.0,CH3Cl);1H NMR(400MHz,Chloroform-d)δ=7.42–7.35(m,2H,Ar),7.12(m,J=7.8,2H,Ar),5.20(dd,J=9.4,9.4Hz,1H,H-3),5.02(dd,J=9.4,9.8Hz,1H,H-4),4.93(dd,J=10.0,9.4Hz,1H,H-2),4.63(d,J=10.0Hz,1H,H-1),4.25-4.13(m,2H,H-6a,6b),3.70(ddd,J=9.8,4.8,2.7Hz,1H,H-5),2.35(s,3H,SPhCH3),2.09(s,3H,OAc),2.08(s,3H,OAc),2.01(s,3H,OAc),1.98(s,3H,OAc).13C NMR(101MHz,CDCl3)δ=170.6,170.2,169.4,169.2,
138.8,133.8,129.7,127.5,85.8(C-1),75.8,74.0,69.9,68.2,62.1,21.2,20.8,20.7,20.6,20.6;HRMS(ESI)m/z calcd for C21H26O9SNa[M+Na]+477.1190,found 477.1198.
化合物3:将化合物2(7.3g,16.1mmol)溶于甲醇(108mL)。加入MeONa(173mg,3.2mmol),反应在室温下搅拌1h。反应液用IR-120树脂(H+)中和。过滤浓缩,粗产品与甲苯共沸2次。将得到的粗产物溶解在DMF(54mL)中,加入对甲基苯甲醛二甲缩醛(3.6mL,24.1mmol)和一水合对甲苯磺酸(456mg,2.4mmol),反应在65℃下搅拌12h,用NEt3淬灭反应。浓缩后用柱层析纯化(石油醚/乙酸乙酯,1:1),得到化合物3(5.4g,89%)。[α]22
D=25.6(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ=7.50–7.41(m,4H,Ar),7.38–7.32(m,3H,Ar),7.16–7.11(m,2H,Ar),5.51(s,1H,ArCH),4.54(d,J=9.7Hz,1H,H-1),4.36(m,1H,H-6a),3.87–3.67(m,2H,H-6b,H-4),3.52–3.44(m,2H,H-3,H-2),3.41(m,1H,H-5),2.93(d,J=2.2Hz,1H,OH),2.75(d,J=2.4Hz,1H,OH),2.36(s,3H,SPhCH3);13C NMR(101MHz,CDCl3)δ=138.9,136.9,133.7,129.9,129.3,128.3,127.2,126.3,101.9,88.7(C-1),80.2,74.5,72.5,70.5,68.6,21.2.HRMS(ESI)m/z calcd for C20H22O5SNa[M+Na]+397.1080,found 397.1078.
化合物4:将化合物3(498mg,1.33mmol)和Bu2SnO(397mg,1.6mmol)溶解在无水甲苯(4.3mL)中加热回流6个小时。混合物冷却到室温,浓缩。粗产物溶解在DMF(6.7mL)中,然后加入CsF(260mg,1.73mmol),TBAB(560mg,1.73mmol),2-NapBr(350mg,1.6mmol),反应液在70℃下搅拌12h。原料反应完后减压浓缩,溶解于二氯甲烷,一次用水和饱和食盐水洗涤。用无水MgSO4干燥,浓缩,柱层析纯化(石油醚/乙酸乙酯,5:1)得到化合物4(417mg,61%)。[α]22
D=-33.8(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.89–7.59(m,4H,Ar),7.51–7.41(m,7H,Ar),7.39–7.34(m,3H,Ar),7.12(m,J=7.9Hz,2H,Ar),5.57(s,1H,ArCH),5.09(d-like,J=11.8Hz,1H,ArCH),4.96(d-like,J=11.8Hz,1H,ArCH),4.55(d,J=9.7Hz,1H,H-1),4.37(dd,J=10.5,4.9Hz,1H,H-6a),3.78(dd,J=10.5,10.5Hz,1H,H-6b),3.66(m,1H,H-4),3.66(dd,J=9.2,9.2Hz,1H,H-3),3.56–3.42(m,2H,H-2,H-5),2.57(s,1H,OH),2.34(s,3H,SPhCH3).13C NMR(101MHz,CDCl3)δ=138.8,137.2,135.7,133.8,133.3,133.1,129.8,129.0,128.3,128.2,127.9,127.7,127.2,126.9,126.1,126.0,126.0,125.9,101.4,88.7(C-1),81.5,81.1,74.8,72.3,70.8,68.7.HRMS(ESI)m/z calcd for C31H30O5SNa[M+Na]+537.1706,found 537.1701.
化合物5:将化合物4(130mg,0.25mmol)溶解在无水DMF(1.4mL),溶液降温到0℃。
加入NaH(22.4mg,0.56mmol),然后加入BnBr(49.9uL,0.42mmol),反应液在室温下搅拌3.5h。待反应物消失后,溶液冷却到0℃,逐滴加入水淬灭反应。反应液用DCM萃取3次,有机相用无水Na2SO4干燥,浓缩。柱层析分离纯化(石油醚/乙酸乙酯,6:1),得到化合物5(130mg,86%)。[α]22
D=-9.3(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.86–7.62(m,4H,Ar),7.52–7.27(m,15H,Ar),7.11(d,J=7.9Hz,2H,Ar),5.60(s,1H,ArCH),5.08(d-like,J=11.4Hz,1H,ArCH),4.93(d-like,J=11.4Hz,1H,ArCH),4.90(d-like,J=10.4Hz,1H,ArCH),4.83(d-like,J=10.4Hz,1H,ArCH),4.70(d,J=9.8Hz,1H,H-1),4.38(dd,J=10.3,5.0Hz,1H,H-6a),3.88(dd,J=9.4,8.3Hz,1H,H-3),3.80(dd,J=10.3,9.8Hz,1H,H-6b),3.71(dd,J=9.4,9.4Hz,1H,H-4),3.51(dd,J=9.8,8.3Hz,1H,H-2),3.45(ddd,J=9.7,9.8,4.9Hz,1H,H-5),2.33(s,3H,SPhCH3);13C NMR(101MHz,CDCl3)δ138.2,138.1,137.3,135.8,133.3,133.0,133.0,129.8,129.2,129.0,128.4,128.3,128.2,128.1,127.9,127.8,127.6,126.8,126.2,126.0,126.0,125.8,101.2,88.6(C-1),83.0,81.5,80.5,75.8,75.3,70.2,68.7,21.1.HRMS(ESI)m/z calcd for C38H36O5SNa[M+Na]+627.2176,found 627.2171.
化合物5’:化合物5(768mg,1.1mmol)溶解在DCM/H2O(9:1,v/v,48.9mL)的混合溶剂中,反应液冷却至0℃,加入2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)(186mg,0.82mmol),反应温度升高至室温继续反应4h。反应液用DCM稀释,用溶液依次用10%的Na2S2O3溶液,饱和的NaHCO3溶液,Na2SO4干燥,过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯,10:1)得到化合物5’(275mg,92%),结构鉴定数据已有文献报道。
化合物6:将化合物(455mg,0.75mmol)溶解在干燥的DCM(3.75mL)中。溶液冷却到0℃,加入BH3·THF(1M,3.75mL),在0℃下搅拌10分钟。逐滴加入TMSOTf(20.3μL,0.11mmol),反应液恢复到室温搅拌1h。原料消失后,加入饱和的NaHCO3终止反应。混合液用DCM萃取3次。用无水Na2SO4干燥,浓缩,过滤。柱层析分离纯化(石油醚/乙酸乙酯,5:1)得到化合物5(441mg,97%)。[α]22
D=17.1(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.86–7.62(m,4H,Ar),7.51–7.35(m,6H,Ar),7.36–7.24(m,8H,Ar),7.12(d,J=7.9Hz,2H,Ar),5.06(d-like,J=11.2Hz,1H,ArCH),5.01(d-like,J=11.2Hz,1H,ArCH),4.95(d-like,J=10.3Hz,1H,ArCH),4.87(d-like,J=11.0Hz,1H,ArCH),4.78(d-like,J=10.3Hz,1H,ArCH),4.67(d-like,1H,11.0Hz,ArCH),4.66(d,J=9.8Hz,1H,H-1),3.88(ddd,J=12.0,6.3,2.7Hz,1H,H-6a),3.77(dd,J=9.1,8.9Hz,1H,H-3),3.70(ddd,J=12.0,7.3,4.9Hz,1H,H-6b),3.59(dd,J=9.1,9.5Hz,1H,H-4),3.49(dd,J=9.8,8.9Hz,1H,H-2),3.38(ddd,J=9.5,4.9,2.7Hz,1H,H-5),2.34(s,3H,SPhCH3),1.91(dd,J=6.3,7.3Hz,1H,OH).13C NMR(101MHz,CDCl3)δ138.1,138.0,137.9,135.8,133.3,133.0,132.7,129.8,129.5,128.5,
128.4,128.2,128.0,127.9,127.9,127.9,127.7,126.5,126.1,125.9,125.8,87.8(C-1),86.6,81.2,79.3,77.7,75.8,75.5,75.1,62.2,21.1.HRMS(ESI)m/z calcd for C38H38O5SNa[M+Na]+629.2332,found 629.2324.
化合物7:将化合物5(194mg,0.33mmol)溶解在吡啶(3.2mL)中,在0℃搅拌5min。加入BzCl(74μL,0.64mmol)和催化量的DMAP。反应液在室温下搅拌4h。反应液一次用饱和NaHCO3,饱和食盐水洗涤。无水Na2SO4干燥,浓缩。柱层析纯化(石油醚/乙酸乙酯,5:1)得到化合物7(224mg,98%)。[α]22
D=9.1(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.13–7.85(m,2H,Ar),7.87–7.65(m,4H,Ar),7.65–7.50(m,1H,Ar),7.52–7.38(m,9H,Ar),7.37–7.29(m,3H,Ar),7.28–7.17(m,5H,Ar),7.04–6.66(m,2H,Ar),5.08(d-like,J=11.0Hz,1H,ArCH),5.01(d-like,J=11.0Hz,1H,ArCH),4.97(d-like,J=10.3Hz,1H,ArCH),4.90(d-like,J=10.8Hz,1H,ArCH),4.77(d-like,J=10.3Hz,1H,ArCH),4.68(dd,J=11.9,1.6Hz,1H,H-6a),4.64(d-like,J=10.8Hz,1H,ArCH),4.64(d,J=9.7Hz,1H,H-1),4.46(dd,J=11.9,4.5Hz,1H,H-6b),3.81(dd,J=8.8,8.8Hz,1H,H-3),3.70-3.63(m,2H,H-4,H-5),3.52(dd,J=9.7,8.8Hz,1H,H-2),2.26(s,3H,SPhCH3);13C NMR(101MHz,CDCl3)δ166.1,138.1,137.9,137.6,135.7,133.3,133.1,133.1,133.0,130.0,129.8,129.6,129.2,128.5,128.5,128.4,128.3,128.2,128.1,128.0,127.9,127.9,127.7,126.7,126.1,126.0,125.9,87.5(C-1),86.8,80.8,77.7,76.1,75.5,75.3,75.2,63.6,21.1.HRMS(ESI)m/z calcd for C45H42O6SNa[M+Na]+733.2594,found 733.2589.
化合物8:将化合物7(100mg,0.14mmol)溶解于DCM/H2O(v/v,10:1,7mL),将溶液冷却到0℃,加入TfOH(1.2uL,0.014mmol)。反应在0℃下搅拌3h。加入NEt3终止反应。反应液用饱和的NaHCO3洗涤,无水Na2SO4干燥,浓缩。柱层析纯化得到中间产物(78.6mg)。把中间产物溶于干燥的DCM(3.75ml)中,反应液冷却到0℃,加入N-苯基三氟乙酰氯(97μl,0.365mmol)和DBU(58μl,0.39mmol)。反应在室温下搅拌2h,浓缩反应液,柱层析纯化(石油醚/乙酸乙酯,20:1)得到化合物8(96mg,88%)。[α]22
D=71.3(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.14–8.00(m,5H),7.94–7.73(m,12H),7.68–7.07(m,37H),6.87–6.67(m,5H),6.55(bs,1H,H-1α),5.80(bs,1H,H-1β),5.27–4.80(m,13H,ArCH),4.75–4.55(m,8H,ArCH,H-6α,H-6β),4.26-4.12(m,2H),3.94–3.63(m,8H).13C NMR(101MHz,CDCl3)δ166.2,166.1,143.5,143.4,137.7,137.6,137.5,137.4,135.8,135.6,133.4,133.3,133.2,133.2,133.1,129.8,129.7,129.1,128.8,128.7,128.6,128.6,128.6,128.5,128.4,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.0,128.0,128.0,127.8,127.7,127.7,126.9,126.7,126.2,126.2,126.1,126.1,126.0,126.0,125.9,125.3,124.4,
124.2,119.4,119.3,97.0(C-1β),93.0(C-1α),84.5,81.6,80.8,79.5,77.3,76.1,75.9,75.5,75.5,75.3,75.2,73.9,73.4,71.7,63.2,63.0,21.5.HRMS(ESI)m/z calcd for C46H40F3NO7Na[M+Na]+798.2649。
实施例2
糖砌块21的合成,路线为图2。
如图2所示,以2,3-O-丙叉基-4-O-卞基甘露乙硫糖苷9为起始原料,经过swern氧化,得到相应的醛化合物10。然后经过witting延长6位的碳链,得到6位脱氧的烯烃化合物11。烯烃化合物在锇酸钾(K2OsO4),铁氰酸钾(K3Fe(CN)6)和碳酸钾(K2CO3)的共同作用下进行双羟基化,得到6,7-二-羟基化合物12。在7-OH上利用二丁基氧化锡(Bu2SnO)选择性的用Nap保护化合物13。6-OH用Bn保护得到化合物14。在80%的醋酸作用下脱去丙叉基后得到化合物15,选择性的利用Nap保护3-OH,得到庚糖砌块16。糖砌块16的2-OH用Lev保护后得到化合物17。化合物17与五碳连接臂(linker)反应,得到端基位连接臂的庚糖18。7-Nap在DDQ的作用下离去得到化合物19。化合物的7-OH用乙酰基保护得到化合物20,最后在醋酸肼的作用下脱去Lev得到庚糖砌块21。
具体试验操作和步骤:
化合物11:将草酰氯(0.36mL,4.23mmol)溶解在干燥的DCM(2.2mL)中,溶液温度降至-78℃,把溶解于DCM(3mL)中的DMSO(0.6mL,8.46mmol)逐滴加入到草酰氯溶液中,反应在-78℃中搅拌0.5h。将化合9(1.0g,2.82mmol)溶解在DCM(11.5mL)中逐滴加入到反应液中。反应液在-78℃搅拌1h,加入NEt3,反应温度在4h内逐渐恢复到室温。待原料反应完毕,加入水终止反应。反应液用DCM萃取,有机相依次用水,饱和食盐水洗涤。无水Na2SO4干燥,浓缩。将甲基三苯基溴化磷溶解到THF中,溶液在0℃搅拌,n-BuLi(4.7mL,11.3mmol,2.4MinTHF)加入进反应瓶,反应搅拌30min后,反应温度降低到-78℃,将上一步粗产物溶解到THF(5mL)中,逐滴加入到反应液中,反应逐渐恢复到室温,继续反应12h。加入饱和的NH4Cl终止反应,反应液用乙酸乙酯萃取3次,用无水年Na2SO4干燥,浓缩。柱层析纯化(石油醚/乙酸乙酯,20:1)得到化合物11(592mg,60%)。[α]22
D=129.25(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.39–7.26(m,5H,Ar),5.99(ddd,J=17.3,10.6,5.5Hz,1H,H-6),5.58(s,1H,H-1),5.41(ddd,J=17.3,1.6,1.6Hz,1H,H-7a),5.25(ddd,J=10.6,1.6,1.6Hz,1H,H-7b),4.85(d-like,J=11.6Hz,1H,ArCH),4.63(d-like,J=11.6Hz,1H,ArCH),4.42(dddd,J=
10.0,5.5,1.6,1.6Hz,1H,H-5),4.29(dd,J=7.2,5.7Hz,1H,H-3),4.19(d,J=5.7Hz,1H,H-2),3.38(dd,J=10.0,7.2Hz,1H,H-4),2.72–2.39(m,2H,SCH2CH3),1.49(s,3H,CH3),1.36(s,3H,CH3),1.28(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ138.2,134.9,128.2,128.0,127.6,117.3,109.4,80.1(C-1),79.5,78.5,73.2,69.5,28.0,26.4,24.4,14.6.HRMS(ESI)m/z calcd for C19H26O4SNa[M+Na]+373.1449,found 373.1447.
化合物12:将K2OsO2(OH)4(103mg,0.28mmol),K3Fe(CN)6(11.2g,33.9mmol)和K2CO3(5.2g,37.3mmol)加入到含有叔丁醇(56.5mL)和水(56.5mL)的反应瓶中。,反应液冷却到0℃,将化合物11溶解在甲苯(22mL)中,加入到反应瓶中,反应在0℃下搅拌29h,待原料完全消失,反应用乙酸乙酯萃取,有机相用1MKOH洗涤。无水Na2SO4干燥,过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯,1:1)得到化合物12(3.2g,74%)。[α]22
D=173.6(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.43–7.28(m,5H,Ar),5.55(s,1H,H-1),4.98(d-like,J=11.4Hz,1H,ArCH),4.64(d-like,J=11.4Hz,1H,ArCH),4.32(dd,J=7.0,5.7Hz,1H,H-3),4.20(d,J=5.7Hz,1H,H-2),4.06(dd,J=9.9,6.3Hz,1H,H-5),3.88(dt,J=6.3,4.2Hz,1H,H-6),3.69(dd,J=9.9,7.0Hz,1H,H-4),3.65(m,2H,H-7a,H-7b),3.54(bs,1H,OH),2.80–2.47(m,2H,SCH2CH3),2.27(bs,1H,OH),1.54(s,3H,CH3),1.37(s,3H,CH3),1.29(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ137.2,128.6,128.3,128.2,109.6,79.6(C-1),79.5,78.3,76.5,73.2,72.8,67.7,62.8,28.1,26.3,24.1,14.1.HRMS(ESI)m/z calcd for C19H28O6SNa[M+Na]+407.1499,407.1491.
化合物13:将化合物12(11.2mg,29.3mmol)溶解到甲苯(146mL)中,在反应瓶中加入Bu2SnO(10.9g,44mmol),反应液加热回流4h。蒸掉一半的甲苯,反应液冷却到室温。加入CsF(6.7g,44mmol),2-NapBr(9.7g,44mmol),CH3CN(73.3mL)。反应液在70℃下反应过夜。反应液用硅藻土过滤,浓缩。柱层析分离纯化(石油醚/乙酸乙酯,3:1)得到化合物12(3.2g,74%)。[α]22
D=78.4(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.83–7.75(m,4H,Ar),7.47–7.33(m,3H,Ar),7.33–7.26(m,5H,Ar),5.51(s,1H,H-1),4.91(d-like,J=11.4Hz,1H,ArCH),4.70(d-like,J=12.0Hz,1H,ArCH),4.65(d-like,J=12.0Hz,1H,ArCH),4.59(d-like,J=11.3Hz,1H,ArCH),4.30(dd,J=6.6,5.8Hz,1H,H-3),4.17(dd,J=5.8,1H,H-2),4.12(m,1H,H-6),4.09(dd,J=9.6,4.8Hz,1H,H-5),3.70(dd,J=9.6,6.6Hz,1H,H-4),3.61(m,2H,H-7a,H-7b),3.02(d,J=2.9Hz,1H,OH),2.70–2.37(m,2H,SCH2CH3),1.51(s,3H,CH3),1.36(s,3H,CH3),1.21(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ137.7,135.7,133.3,133.0,128.4,128.4,128.1,128.1,127.9,127.8,127.7,126.4,126.0,125.8,125.7,109.5,79.7(C-1),78.6,78.0,76.7,76.5,73.6,72.8,72.3,71.0,68.5,28.0,26.4,24.1,14.2.
HRMS(ESI)m/z calcd for C30H36O6SNa[M+Na]+547.2125,found 547.2135.
化合物14:将化合物13(10.3g,19.6mmol)溶解在无水DMF(1.4mL),溶液降温到0℃加入NaH(1.56g,39.2mmol),然后加入BnBr(4.7mL,39.2mmol),反应液在室温下搅拌3.5h。待反应物消失后,溶液冷却到0℃,逐滴加入水淬灭反应。反应液用DCM萃取3次,有机相用无水Na2SO4干燥,浓缩。柱层析分离纯化(石油醚/乙酸乙酯,10:1),得到化合物14(11.0g,91%)。1H NMR(400MHz,Chloroform-d)δ7.83–7.69(m,4H,Ar),7.49–7.24(m,13H,Ar),5.55(s,1H,H-1),4.82(d-like,J=11.4Hz,1H,ArCH),4.74(d-like,J=11.8Hz,1H,ArCH),4.69(d-like,J=11.8Hz,1H,ArCH),4.65(d-like,J=12.3Hz,1H,ArCH),4.59(d-like,J=12.3Hz,1H,ArCH),4.53(d-like,J=11.4Hz,1H,ArCH),4.33–4.23(m,2H,H-3,H-5),4.14(d,J=5.6Hz,1H,H-2),4.08(ddd,J=5.8,5.3,1.5Hz,1H),3.75–3.72(m,2H,H-7a,H-7b),3.70(dd,J=10.3,6.9Hz,1H,H-4),2.77–2.41(m,2H,SCH2CH3),1.41(s,3H,CH3),1.34(s,3H,CH3),1.22(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ138.7,138.2,136.0,133.3,132.9,128.2,128.2,128.0,127.9,127.8,127.7,127.6,127.5,127.4,126.2,126.0,125.7,125.6,109.3,79.5(C-1),79.0,77.8,76.4,76.3,73.2,72.7,72.5,70.5,69.3,28.0,26.5,23.8,14.3.HRMS(ESI)m/z calcd for C37H42O6SNa[M+Na]+637.2594,found 637.2587.
化合物15:将化合物14溶解到80%的醋酸水溶液中,反应液在60℃度搅拌12h。TLC板显示原料反应完。反应液浓缩后用DCM稀释,饱和NaHCO3洗涤,Na2SO4干燥,过滤浓缩,柱层析分离纯化(石油醚/乙酸乙酯,3:1),得到化合物16(9.8g,96%)。[α]22
D=71.5(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.92–7.70(m,4H,Ar),7.50–7.27(m,8H,Ar),7.25–7.15(m,5H,Ar),5.26(d,J=1.9Hz,1H,H-1),4.76(d-like,J=11.9Hz,1H,ArCH),4.71(d-like,J=11.9Hz,1H,ArCH),4.70–4.61(m,4H,ArCH),4.30(dd,J=9.4,1.6Hz,1H,H-5),4.03(ddd,J=6.8,5.2,1.6Hz,1H,H-6),3.94(dd,J=3.4,1.9Hz,1H,H-2),3.89(dd,J=8.6,3.3Hz,1H,H-3),3.84(dd,J=10.2,5.2Hz,1H,H-7a),3.84(dd,J=9.4,8.6Hz,1H,H-4),3.74(dd,J=10.2,6.8Hz,1H,H-7b),2.75–2.49(m,2H,SCH2CH3),2.45(bs,1H,OH),2.23(bs,1H,OH),1.24(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ138.7,138.3,135.8,133.4,133.1,128.7,128.4,128.3,128.1,128.0,127.9,127.8,127.7,126.6,126.2,126.0,125.9,83.5(C-1),77.8,76.5,74.4,73.6,72.7,72.6,72.2,71.7,70.6,24.8,14.9.HRMS(ESI)m/z calcd for C34H38O6SNa[M+Na]+597.2281,found 597.2278.
化合物16:将化合物15(50mg,87umol)溶解到甲苯(0.58mL)中,在反应瓶中加入Bu2SnO(24.9mg,0.1mmol),反应液加热回流3h。反应液冷却到室温。加入CsF(15.2mg,
0.1mmol),TBAB(32.2mg,0.1mmol),BnBr(11.9化合物μL,44mmol)。反应液加热回流三个小时。反应液用硅藻土过滤,浓缩。柱层析分离纯化(石油醚/乙酸乙酯,3:1)得到化合物16(43mg,74%)。[α]22
D=87.2(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.85–7.67(m,4H),7.49–7.35(m,5H),7.34–7.07(m,13H),5.34(d,J=1.5Hz,1H,H-1),4.96–4.53(m,8H,ArCH),4.32(dd,J=9.7,1.3Hz,1H,H-5),4.06(dd,J=3.4,1.8Hz,1H,H-2),4.04–4.01(m,1H,H-6),3.92(dd,J=9.7,9.1Hz,1H,H-4),3.85(dd,J=9.1,3.2Hz,1H,H-3),3.80(dd,J=10.3,4.7Hz,1H,H-7a),3.74(dd,J=10.3,6.9Hz,1H,7H-b),2.77–2.26(m,3H,SCH2CH3,OH),1.24(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ138.7,138.7,138.3,138.2,137.5,137.5,135.9,135.9,133.3,132.9,128.6,128.6,128.3,128.3,128.2,128.2,128.1,128.0,128.0,128.0,128.0,127.9,127.8,127.8,127.7,127.6,127.6,127.5,127.4,127.4,126.2,126.0,126.0,125.7,125.7,83.0(C-1),80.9,78.0,74.7,74.6,73.3,72.3,72.1,71.8,70.8,69.6,69.6,24.6,14.7.HRMS(ESI)m/z calcd for C41H44O6SNa[M+Na]+687.2751,found 687.2753.
化合物17:化合物16(728mg,1.1mmol)溶解于DCM(24mL),加入LevOH(192mg,1.65mmol),二环己基碳亚胺(DCC)(340mg,1.65mmol),N,N-二甲基吡啶(DMAP)(202mg,1.65mmol),反应在室温下搅拌2h。浓缩,柱层析分离纯化(石油醚/乙酸乙酯,3:1)得到化合物17(839mg,100%)。[α]22
D=54.8(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.85–7.71(m,4H,Ar),7.50–7.36(m,5H,Ar),7.35–7.22(m,11H,Ar),7.15(m,2H,Ar),5.40(dd,J=2.9,1.7Hz,1H,H-2),5.25(d,J=1.7Hz,1H,H-1),4.85–4.57(m,8H,ArCH),4.31(d,J=9.1Hz,1H,H-5),4.04(ddd,J=6.9,4.6,1.2Hz,1H,H-6),3.95(dd,J=9.1,9.1Hz,1H,H-4),3.91(dd,J=9.1,2.8Hz,1H,H-3),3.79(dd,J=10.4,4.6Hz,1H,H-7a),3.73(dd,J=10.4,6.9Hz,1H,H-7b),2.82–2.35(m,6H,SCH2CH3,C(O)CH2CH2(O)C),2.09(s,3H,CH3),1.24(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ206.2,171.9,138.7,138.3,137.6,135.9,133.2,132.9,128.4,128.3,128.3,128.2,128.2,128.1,128.0,127.9,127.8,127.8,127.8,127.6,127.5,127.5,127.4,126.2,126.1,126.0,125.7,125.6,82.0(C-1),78.9,78.4,76.7,74.8,74.5,73.3,72.3,72.2,71.6,71.0,70.4,37.9,29.7,28.0,25.3,14.8.HRMS(ESI)m/z calcd for C46H50O8SNa[M+Na]+785.3119,found785.3106.
化合物18:化合物17(595mg,0.78mmol)和N-卞基-N-苯甲氧羰基-5-氨基-戊醇(510mg,0.78mmol)用甲苯共蒸三次,混合物中加入干燥的DCM(5mL),4
分子筛。混合液在室温下搅拌半个小时后冷却到0℃,加入NIS(211mg,0.94mmol)和TMSOTf(14μL,78ummol),反应液在0℃下搅拌3h。加入NEt
3终止反应。过滤,溶液依次用10%的Na
2S
2O
3
溶液,饱和的NaHCO
3溶液,Na2SO4干燥,过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯,2:1)得到化合物18(562mg,72%)。[α]
22
D=11.5(c1.0,CH
3Cl).
1H NMR(400MHz,Chloroform-d)δ7.90–7.62(m,4H,Ar),7.53–7.29(m,21H,Ar),7.28–7.11(m,7H,Ar),5.34(d,J=3.1Hz,1H,H-2),5.20(d,J=11.0Hz,2H,COOCH
2Ph),4.87(d-like,J=10.6Hz,1H,ArCH),4.83-4.74(m,3H,H-1,ArCH),4.72–4.60(m,7H,ArCH),4.08(dd,J=6.7,4.6Hz,1H,H-5),3.99-3.90(m,3H,H-3,H-4,H-6),3.83(dd,J=10.4,4.6Hz,1H,H-7a),3.78(dd,J=10.4,6.7Hz,1H,H-7b),3.63(m,1H,OCH
2),3.37–3.16(m,3H,OCH
2,NCH
2),2.75–2.51(m,4H,C(O)CH
2CH
2(O)C),2.11(s,1H,CH
3),1.66–1.45(m,4H,CH
2CH
2),1.31–1.11(m,2H,CH
2);
13C NMR(101MHz,CDCl
3)δ206.3,172.0,138.8,138.3,138.0,135.9,133.3,132.9,128.6,128.5,128.4,128.4,128.3,128.3,128.1,128.1,128.0,127.9,127.9,127.8,127.7,127.6,127.6,127.4,127.2,126.2,126.1,125.8,125.6,97.4(C-1),78.7,78.3,75.0,74.4,73.4,72.5,72.0,71.7,70.9,68.8,67.6,67.2,50.5,50.2,47.1,46.1,37.9,29.8,29.1,28.1,28.0,27.5,23.4.HRMS(ESI)m/z calcd for C
64H
69NO
11Na[M+Na]
+1050.4763,found 1050.4736.
化合物19:化合物18(420mg,0.41mmol)溶解在DCM/H2O(9:1,v/v,15.2mL)的混合溶剂中,反应液冷却至0℃,加入2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)(186mg,0.82mmol),反应温度升高至室温继续反应三个小时。反应液用DCM稀释,用溶液依次用10%的Na2S2O3溶液,饱和的NaHCO3溶液,Na2SO4干燥,过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯,1:1)得到化合物19(275mg,76%)。[α]22
D=13.8(c0.5,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.48–7.18(m,25H,Ar),5.35(s,1H,H-2),5.20(d,J=15.5Hz,2H,COOCH2Ph),4.93(d-like,J=10.7Hz,1H,ArCH),4.78(bs,2H,H-1,ArCH),4.74–4.46(m,6H,ArCH),4.02–3.90(m,2H,H-6,H-5),3.84(m,3H,H-3,H-4,H-7a),3.65(m,3H,H-7b,OCH2),3.45–3.14(m,3H,OCH2,NCH2),2.87–2.61(m,4H,C(O)CH2CH2(O)C),2.17(s,3H,CH3),1.74–1.46(m,4H,CH2CH2),1.32–1.17(m,2H,CH2);13C NMR(101MHz,CDCl3)δ172.0,137.9,137.8,128.6,128.5,128.4,128.4,128.2,128.1,128.0,127.9,127.8,127.7,127.3,127.2,97.3(C-1),78.6,75.0,74.2,72.0,71.9,71.7,68.7,67.9,67.3,50.5,50.2,47.1,46.1,38.1,29.9,29.1,28.2,28.0,27.5,23.4.HRMS(ESI)m/z calcd for C53H61NO11Na[M+Na]+910.4137,found 910.4117.
化合物20:将化合物19(210mg,0.24mmol)溶解于吡啶(2.3mL)中,在0℃下,加入醋酸酐(0.45mL,4.74mmol)以及催化量的DMAP,反应液恢复到室温,搅拌12h,浓缩,柱层析分离纯化(石油醚/乙酸乙酯,3:1)得到化合物20(203mg,92%)。[α]22
D=20.4(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.51–7.04(m,25H,Ar),5.35(d,J=3.1
Hz,1H,H-2),5.21(d,J=14.5Hz,1H,COOCH2Ph),4.93(d-like,J=10.7Hz,1H),4.78–4.71(m,3H,H-1,ArCH),4.67–4.47(m,5H,ArCH),4.34(dd,J=12.1,3.5Hz,1H,H-7a),4.27(dd,J=12.0,7.8Hz,1H,H-7b),4.02(dd,J=8.9,4.0Hz,1H,H-5),3.95(dd,J=7.8,3.5Hz,1H,H-6),3.92(m,1H,H-3),3.83(dd,J=8.9,8.9Hz,1H,H-4),3.64(m,1H,OCH2),3.44–3.12(m,3H,OCH2,NCH2),2.84–2.64(m,4H,C(O)CH2CH2(O)C),2.19(s,3H,CH3),2.02(s,3H,OAc),1.62–1.47(m,4H,CH2CH2),1.38–1.18(m,2H,CH2);13C NMR(101MHz,CDCl3)δ206.3,172.0,170.8,138.3,138.1,137.9,137.9,128.6,128.5,128.5,128.4,128.4,128.3,128.2,128.1,128.1,128.0,127.9,127.9,127.9,127.8,127.8,127.7,127.6,127.4,127.3,127.2,97.4(C-1),78.6,76.6,75.0,74.1,72.1,71.7,71.7,71.7,68.6,67.7,67.2,64.6,50.6,50.2,47.1,46.1,38.0,29.9,29.8,29.2,28.2,27.9,27.5,23.4,23.4,21.0.HRMS(ESI)m/z calcd for C55H63NO12Na[M+Na]+952.4242,found 952.4233.
化合物21:将化合物20(190mg,0.2mmol)溶解在DCM/MeOH(20:1,v/v,4mL)混合溶剂中,加入醋酸肼(27.6mg,0.3mmol),反应液在室温下搅拌2h,反应液用NaHCO3洗涤,用无水Na2SO4干燥。浓缩柱层析纯化分离(石油醚/乙酸乙酯,3:1)得到化合物21(158mg,95%)。[α]22
D=34.5(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.45–7.13(m,25H),5.18(d,J=14.0Hz,2H,COOCH2Ph),4.87(d-like,J=10.8Hz,1H,ArCH),4.81(m,1H,H-1),4.76–4.45(m,7H,ArCH),4.31(dd,J=12.1,3.8Hz,1H,H-7a),4.25(dd,J=12.1,7.6Hz,1H,H-7b),4.00(s,1H,H-2),3.94–3.85(m,3H,H-3,H-5,H-6),3.82(dd,J=9.6,8.4Hz,1H,H-4),3.69–3.56(m,1H,OCH2),3.37–3.16(m,3H,OCH2,NCH2),2.42(bs,1H,OH),1.97(s,3H,OAc),1.63–1.64(m,4H,CH2CH2),1.37–1.13(m,2H,CH2);13C NMR(101MHz,CDCl3)δ170.8,138.3,138.1,137.9,137.8,128.6,128.6,128.5,128.4,128.3,128.3,128.2,128.2,128.1,128.0,128.0,127.9,127.9,127.9,127.7,127.6,127.3,127.2,98.9(C-1),80.8,76.4,75.0,74.1,72.1,72.0,71.4,68.1,67.5,67.2,64.4,50.5,50.2,47.1,46.1,29.1,27.9,27.5,23.4,21.0.HRMS(ESI)m/z calcd for C50H57NO10Na[M+Na]+854.3875,found 854.3863.
实施例3
糖砌块28的合成,路线为图3。
如图3所示,以化合物22起始原料,2-OH用Bz保护,得到化合物23。化合物23在80%醋酸作用下打开丙叉基,得到化合物24。化合物24的3-OH选择性的用Bn保护得到化合物25。化合物25的4-OH用Lev保护,得到化合物26。硫糖苷26在NBS条件
下水解,裸露端基位OH,端基位-OH与叔丁基二甲基氯硅烷(TBSCl)反应,得到端基TBS全保护的半乳糖27。在醋酸肼作用下脱去Lev得到半乳糖砌块28。
具体试验操作和步骤:
化合物23:将化合物22(4.2g,11.8mmol)溶解在吡啶(59.5mL)中,在0℃搅拌5min。加入BzCl(2.78mL,24mmol)和催化量的DMAP。反应液在室温下搅拌4h。反应液一次用饱和NaHCO3,饱和食盐水洗涤。无水Na2SO4干燥,浓缩。柱层析纯化(石油醚/乙酸乙酯,10:1)得到化合物23(5.2g,96%)。[α]22
D=22.9(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.10–7.94(m,2H,Ar),7.60–7.51(m,1H,Ar),7.45–7.42(m,2H,Ar),7.38–7.27(m,5H,Ar),5.28(dd,J=9.9,6.8Hz,1H,H-2),4.66(d-like,J=12.0Hz,1H,ArCH),4.58(d-like,J=12.0Hz,1H,ArCH),4.53(d,J=9.9Hz,1H,H-1),4.34(dd,J=6.8,5.4Hz,1H,H-3),4.30(dd,J=5.4,2.0Hz,1H,H-4),4.03(dt,J=6.1,2.0Hz,1H,H-5),3.83(d,J=6.1Hz,2H,H-6a,H-6b),2.89–2.57(m,2H,SCH2CH3),1.61(s,3H,CH3),1.36(s,3H,CH3),1.25(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ165.5,138.1,133.1,129.9,129.8,128.4,128.3,127.7,127.7,110.5,82.9(C-1),77.1,75.9,73.9,73.6,72.2,69.4,27.8,26.4,24.3,14.9.HRMS(ESI)m/z calcd for C25H31O6SN[M+NH4]+476.2101,found 476.2104.
化合物24:将化合物23(1.2g,2.6mmol)溶解到80%的醋酸水溶液中(25mL),反应液在60℃度搅拌5h。TLC板显示原料反应完。反应液浓缩后用DCM溶解,饱和NaHCO3洗涤,Na2SO4干燥,过滤浓缩,柱层析分离纯化(石油醚/乙酸乙酯,1:1),得到化合物24(0.98g,90%)。[α]22
D=-19.4(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.11–7.90(m,2H,Ar),7.63–7.51(m,1H,Ar),7.49–7.41(m,2H,Ar),7.39–7.28(m,5H,Ar),5.30(dd,J=9.6,9.6Hz,1H,H-2),4.60(d,J=1.8Hz,2H,ArCH),4.56(d,J=9.6Hz,1H,H-1),4.13(ddd,J=3.6,3.6,1.1Hz,1H,H-4),3.82(m,2H,H-6a,H-6b),3.78(dd,J=9.6,3.6Hz,1H,H-3),3.71(dt,J=5.3,1.1Hz,1H,H-5),3.13(d,J=7.9Hz,1H,OH),3.03(d,J=3.6Hz,1H,OH),2.86–2.63(m,2H,SCH2CH3),1.26(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ166.8,137.7,133.3,130.0,129.7,128.5,128.4,127.9,127.8,83.3(C-1),77.2,73.9,73.8,72.3,69.9,69.6,23.9,15.0.HRMS(ESI)m/z calcd for C22H26O6SNa[M+Na]+441.1342,found 441.1347.
化合物25:将化合物24(4.2g,10.1mmol)溶解到甲苯(60mL)中,在反应瓶中加入Bu2SnO(3.8g,15.2mmol),反应液加热回流2h。反应液冷却到室温。加入TBAI(5.6g,15.2mmol),BnBr(1.8g,15.2mmol)。反应液在65℃下反应过夜。反应液用硅藻土过滤,浓
缩。柱层析分离纯化(石油醚/乙酸乙酯,6:1)得到化合物25(4.5g,87%)。[α]22
D=19.4(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.12–7.89(m,2H,Ar),7.64–7.53(m,1H,Ar),7.45(dd,J=8.3,7.2Hz,2H,Ar),7.40–7.28(m,5H,Ar),7.23–7.12(m,5H,Ar),5.53(dd,J=9.7,9.7Hz,1H,H-2),4.67(d-like,J=12.3Hz,1H,ArCH),4.60(s,2H,ArCH),4.53(d-like,J=12.3Hz,1H,ArCH),4.48(d,J=9.7Hz,1H,H-1),4.19(ddd,J=3.1,1.5,1.5Hz,1H,H-4),3.83(dd,J=9.8,6.4Hz,1H,H-6a),3.77(dd,J=9.8,5.7Hz,1H,H-6b),3.67–3.69(m,1H,H-5),3.65(dd,J=9.7,3.1Hz,1H,H-3),2.81–2.65(m,2H,SCH2CH3),2.64(t,J=1.6Hz,1H),1.22(t,J=7.5Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ165.4,137.9,137.1,133.1,130.0,129.9,128.4,128.4,128.4,128.3,128.0,127.8,127.8,83.5(C-1),79.3,73.8,71.3,69.6,69.0,66.4,23.6,14.9.HRMS(ESI)m/z calcd for C29H32O6SNa[M+Na]+531.1811,found 531.1819.
化合物26:化合物25(4.0g,7.9mmol)溶解于DCM(150mL),加入LevOH(1.36g,11.7mmol),二环己基碳亚胺(DCC)(2.41g,11.7mmol),N,N-二甲基吡啶(DMAP)(1.43mg,11.7mmol),反应在室温下搅拌2h。浓缩,柱层析分离纯化(石油醚/乙酸乙酯,4:1)得到化合物26(3.9g,82%)。[α]22
D=42.1(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.07–7.80(m,2H,Ar),7.64–7.55(m,1H,Ar),7.50–7.41(m,2H,Ar),7.41–7.25(m,5H,Ar),7.22–7.04(m,5H,Ar),5.72(dd,J=3.4,1.1Hz,1H,H-4),5.41(dd,J=9.8,9.8Hz,1H,H-2),4.63(d-like,J=12.7Hz,1H,ArCH),4.54-4.51(m,3H,H-1,ArCH),4.41(d-like,J=12.7Hz,1H,ArCH),3.80(ddd,J=7.0,5.6,1.1Hz,1H,H-5),3.66(dd,J=9.4,5.6Hz,1H,H-6a),3.65(dd,J=9.4,3.0Hz,1H,H-6b),3.56(dd,J=9.4,7.0Hz,1H,H-6b),2.91–2.57(m,6H,SCH2CH3,C(O)CH2CH2(O)C),2.18(s,3H,CH3),1.22(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ206.4,172.1,165.4,137.7,137.3,133.2,130.0,129.8,128.5,128.3,128.3,128.2,128.0,127.9,127.7,83.9(C-1),77.2,76.2,73.8,70.7,69.5,68.0,66.5,38.1,29.9,28.1,24.2,14.9.HRMS(ESI)m/z calcd for C34H38O8SNa[M+Na]+629.2180,found 629.2181.
化合物27:将化合物7(317mg,0.52mmol)溶解于THF/H2O(v/v,1:1,5mL),加入NBS(223mg,1.25mmol)。反应在室温下搅拌5h。反应液用饱和的NaHCO3洗涤,无水Na2SO4干燥,浓缩。柱层析纯化得到中间产物(225mg)。把中间产物溶于干燥的DCM(2.5ml)中,反应液冷却到0℃,加入咪唑(75mg,1.11mmol)和TBSCl(84mg,0.56mmol)。反应在室温下搅拌12h,反应液用饱和NH4Cl淬灭,混合溶液用DCM萃取3遍,有机相用水洗,无水Na2SO4干燥,浓缩,柱层析纯化(石油醚/乙酸乙酯,20:1)得到化合物27(248mg,71%)。[α]22
D=44.8(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.02–7.80(m,2H,Ar),7.64–7.50(m,1H,Ar),7.45(t,J=7.7Hz,2H,Ar),7.39–7.27(m,5H,Ar),7.20–7.04(m,
5H,Ar),5.65(dd,J=3.2,0.8Hz,1H,H-4),5.34(dd,J=10.1,7.7Hz,1H,H-2),4.71(d,J=7.7Hz,1H,H-1),4.63(d-like,J=12.9Hz,1H,ArCH),4.54(s,2H,ArCH),4.40(d-like,J=12.9Hz,1H,ArCH),3.76(t,J=6.2Hz,1H,H-5),3.68–3.61(m,2H,H-6a,H-6b),3.59(dd,J=10.1,3.2Hz,1H,H-3),2.88–2.64(m,4H,C(O)CH2CH2(O)C),2.19(s,3H,CH3),0.75(s,9H,OTBS),0.06(s,3H,OTBS),-0.02(s,3H,OTBS);13C NMR(101MHz,CDCl3)δ206.6,172.1,165.3,137.8,137.5,133.0,130.1,129.8,128.5,128.3,128.2,128.1,127.9,127.8,127.6,96.3(C-1),76.1,73.8,73.1,72.5,70.6,68.2,66.4,38.1,29.9,28.2,25.4,17.8,-4.2,-5.3.HRMS(ESI)m/z calcd for C34H38O9SiNa[M+Na]+699.2960,found 699.2957.
化合物28:将化合物27(203mg,0.30mmol)溶解在DCM/MeOH(20:1,v/v,3.5mL)混合溶剂中,加入醋酸肼(41.4mg,0.45mmol),反应液在室温下搅拌4h,反应液用NaHCO3洗涤,用无水Na2SO4干燥。浓缩柱层析纯化分离(石油醚/乙酸乙酯,6:1)得到化合物28。[α]22
D=18.6(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.00(m,2H,Ar),7.63–7.53(m,1H,Ar),7.49–7.40(m,2H,Ar),7.39–7.26(m,5H,Ar),7.25–7.09(m,5H,Ar),5.42(dd,J=9.8,7.6Hz,1H,H-2),4.71(d,J=7.7Hz,1H,H-1),4.69(d-like,J=12.4Hz,1H,ArCH),4.61(s,2H,ArCH),4.52(d-like,J=12.5Hz,1H),4.13(dd,J=3.4,1.4Hz,1H,H-4),3.87(dd,J=9.8,6.0Hz,1H,H-6a),3.75(dd,J=9.8,6.0Hz,1H,H-6b),3.65(t,J=6.0Hz,1H,H-5),3.59(dd,J=9.8,3.3Hz,1H,H-3),2.60(t,J=1.6Hz,1H,OH),0.74(s,9H,OTBS),0.07(s,3H,OTBS),-0.01(s,3H,OTBS).13C NMR(101MHz,CDCl3)δ165.3,138.1,137.2,132.9,130.2,129.8,128.5,128.4,128.3,127.9,127.9,127.8,96.3(C-1),78.1,73.8,73.6,73.2,71.1,69.2,66.2,25.5,17.8,-4.2,-5.3.HRMS(ESI)m/z calcd for C33H42O7SiNa[M+Na]+601.2592,found 601.2586.
实施例4
糖基化反应的条件和脱去酰基类保护剂条件:
此发明中如未说明,糖基化反应的条件如下:糖基供体和受体在甲苯中共蒸三次。加入干燥的DCM或者DCM/Et
2O(v/v,1:2)反应浓度为0.02-0.05M,活化的3
或者4
分子筛。加入相当于糖基供体10当量的噻酚,混合液在室温下搅拌30min后冷却至0℃,加入TMSOTf为促进剂,反应时间为3-5h。反应结束后用吡啶终止反应。反应液过滤,用DCM稀释后用饱和的NaHCO
3洗涤。无水Na
2SO
4干燥浓缩。柱层析纯化分离。
此发明中如未说明,碱性条件脱去酰基条件为:将起始原料溶解在DCM/MeOH(v/v,
1:1)中,反应浓度为0.05M,加入0.5当量MeONa(5MinMeOH)。反应温度为室温,TLC板显示反应结束用AmerliteIR120(H+)树脂中和反应液pH达到7。过滤,浓缩,柱层析纯化。
实施例5
三糖供体32的组装,路线为图4。
具体试验操作和步骤:
化合物29:根据反应条件4.1,糖基供体8(335mg,0.43mmol)和糖基受体28(301mg,0.52mmol)反应得到29(361mg,72%)。[α]22
D=93.1(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.09–7.94(m,4H,Ar),7.85–7.67(m,4H,Ar),7.63–7.11(m,28H),5.55(dd,J=10.2,7.5Hz,1H,H-2b),5.11(d,J=3.8Hz,3H,H-1a,2×ArCH),4.97(t-like,J=11.3Hz,2H,ArCH),4.77(d,J=7.5Hz,1H,H-1b),4.78–4.73(m,2H,ArCH),4.68(d-like,J=11.2Hz,1H,ArCH),4.62–4.50(m,2H,H-5a,ArCH),4.39(dd,J=9.4,9.4Hz,1H,H-3a),4.33–4.18(m,5H,H-6a,H-6a’,H-4b,2×ArCH),4.10–3.99(m,1H,H-6b),3.79(t,J=9.4Hz,1H,H-4a),3.63(dd,J=10.1,3.9Hz,2H,H-2a),3.60–3.51(m,3H,H-6b’,H-3b,H-5b),0.80(s,9H,OTBS),0.22(s,3H,OTBS),0.10(s,3H,OTBS);13C NMR(101MHz,cdcl3)δ166.02,165.19,138.61,138.22,137.91,137.47,136.04,133.35,132.95,132.82,132.79,130.39,130.17,129.78,129.63,128.37,128.33,128.29,128.25,128.22,128.12,127.99,127.90,127.69,127.67,127.66,127.63,127.60,127.55,127.38,127.05,126.96,126.31,125.89,125.77,99.46(C-1a),96.92(C-1b),81.81,80.84,77.79,77.73,75.90,74.57,74.05,73.77,73.69,73.14,71.44,69.36,67.64,63.08,25.67,17.88,-3.84,-4.27.HRMS(ESI)m/z calcd for C71H76O13SiNa[M+Na]+1187.4947,found 1187.4907.
化合物30:化合物29(940mg,0.81mmol)溶解在DCM/MeOH(9:1,v/v,18.mL)的混合溶剂中,反应液冷却至0℃,加入2,3-二氯-5,6-二氰基-1,4-苯醌(DDQ)(545mg,2.4mmol),反应温度升高至室温继续反应10h。反应液用DCM稀释,用溶液依次用10%的Na2S2O3溶液,饱和的NaHCO3溶液,Na2SO4干燥,过滤浓缩。柱层析分离纯化(石油醚/乙酸乙酯,1:1)得到化合物30(581mg,70%)。[α]22
D=75.6(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.04–7.92(m,4H,Ar),7.60–7.51(m,2H,Ar),7.48–7.27(m,16H,Ar),7.22–7.11(m,8H,Ar),5.46(dd,J=10.3,7.5Hz,1H,H-2b),5.08(d,J=3.4Hz,1H,H-1a),4.86–4.77(m,2H,ArCH),4.71(d,J=7.6Hz,1H,H-1b),4.75–4.68(m,3H,ArCH),4.56-4.48(dd,2H,H-5a,ArCH),4.44(dd,J=9.4,9.4Hz,1H,H-3a),4.24-4.26(d,3H,H-6a,2×ArCH),4.24–4.19(m,2H,H-6a’,H-4b),3.96(dd,J=9.3,7.7Hz,1H,H-6b),3.70–3.61(m,1H,H-4a),
3.62–3.47(m,3H,H-3b,H-5b,H-6b’),3.46(dd,J=10.1,3.4Hz,1H,H-2a),0.76(s,9H,OTBS),0.10(s,3H,OTBS),0.00(s,3H,OTBS);13C NMR(101MHz,CDCl3)δ166.1,165.1,138.4,138.3,137.9,137.4,132.9,132.8,130.3,130.2,129.8,129.7,128.5,128.4,128.4,128.3,128.3,128.1,127.8,127.7,127.6,127.5,98.99(C-1a),96.7(C-1b),80.3,77.8,77.5,73.9,73.8,73.6,73.5,73.3,73.1,72.8,71.4,68.8,67.7,63.2,25.6,17.9,-4.1,-4.9.HRMS(ESI)m/z calcd for C60H68O13SiNa[M+Na]+1047.4321,found 1047.4304.
化合物31:根据反应条件4.1,糖基供体8(100mg,0.13mmol)和糖基受体30(87.3g,0.085mmol)反应得到化合物31(78mg,57%)。[α]22
D=93.4(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.10–7.98(m,8H,Ar),7.85–7.68(m,4H,Ar),7.62–7.04(m,40H,Ar),5.68(d,J=3.5Hz,1H,H-1a),5.55(dd,J=10.3,7.5Hz,1H,H-2c),5.15(d,J=3.1Hz,1H,H-1b),5.13–5.06(m,2H,ArCH),4.99(d-like,J=10.9Hz,1H,ArCH),4.89–4.82(d-like,J=10.9,1H,ArCH),4.80(d,J=7.6Hz,1H,H-1c),4.79–4.62(m,8H,H-3b,H-5b,6×ArCH),4.56–4.48(m,3H,H-5a,2×ArCH),4.47–4.38(m,2H,H-6a,H-6b),4.37–4.23(m,3H,H-6a’,H-3b,ArCH),4.22–4.13(m,3H,H-3a,H-6b’,H-3c),4.03–3.95(m,2H,H-4b,H-6c),3.72(dd,J=10.4,9.2Hz,1H,H-4a),3.67(dd,J=10.1,3.1Hz,1H,H-2a),3.64–3.52(m,4H,H-2b,H-3c,H-5c,H-6c’),0.75(s,9H,OTBS),0.20(s,3H,OTBS),0.09(s,3H,OTBS);13C NMR(101MHz,CDCl3)δ166.1,166.0,165.0,138.3,138.2,138.0,137.9,137.6,137.4,136.1,133.3,132.9,132.9,132.8,132.7,130.4,130.2,130.2,129.8,129.7,129.7,129.1,128.4,128.4,128.4,128.3,128.3,128.3,128.2,128.1,128.0,128.0,127.9,127.9,127.8,127.7,127.7,127.6,127.5,127.2,127.1,126.6,126.1,126.0,125.8,125.3,98.6(C-1b),97.0(C-1a),96.8(C-1c),82.3,80.0,79.9,78.6,78.2,77.7,75.8,75.1,75.0,74.7,73.8,73.6,73.5,73.5,73.2,71.9,71.6,68.7,68.4,68.2,63.2,63.1,25.5,17.8,-3.8,-4.4.HRMS(ESI)m/z calcd for C98H102O19SiNa[M+Na]+1633.6677,found 1633.6667.
化合物32:将化合物31(54.0mg,0.034mmol)溶解于THF(1.7mL),冷却至0℃,加入AcOH(19.4μL,0.34mmol),而后加入1MTBAFinTHF(0.34mL,0.34mmol)。溶液在室温下搅拌2h。反应液用乙酸乙酯稀释后用水洗三次。无水Na2SO4干燥,浓缩,层析柱分离纯化后得到中间产物。中间产物溶解在干燥的DCM中,冷却至0℃,加入N-苯基三氟乙酰氯(20uL,0.14mmol)和DBU(12.6uL,0.084mmol),反应液在室温下搅拌2h。浓缩,层析柱分离纯化得到化合物32(49.3mg,87%)。[α]22
D=122.6(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.08–7.94(m,6H,Ar),7.87–6.52(m,51H,Ar),5.96(bs,1H,H-1c),5.76(dd,J=8.7,8.7Hz,1H,H-2c),5.67(d,J=3.6Hz,1H,H-1a),5.15–5.04(m,3H,H-1b,2×ArCH),4.99(d-like,J=10.9Hz,1H,ArCH),4.85(d-like,J=10.8Hz,1H,ArCH),4.81–4.67(m,
4H,ArCH),4.67–4.47(m,8H,H-5a,H-6a,H-3b,H-5b,4×ArCH),4.47–4.28(m,3H,H-6a’,H-6b’,ArCH),4.28–4.09(m,4H,H-3a,H-6b’,H-4c,ArCH),3.99–3.85(m,2H,H-4b,H-6c),3.82–3.51(m,5H,H-2a,H-4a,H-2b,H-3c,H-5c,H-6c’);13C NMR(101MHz,CDCl3)δ166.1,164.7,138.2,138.1,137.9,137.9,137.5,137.0,136.0,133.3,133.2,133.0,132.9,132.9,130.1,130.0,129.9,129.7,129.6,129.4,128.6,128.5,128.5,128.4,128.4,128.3,128.3,128.3,128.1,128.1,128.0,128.0,128.0,127.9,127.9,127.8,127.8,127.7,127.7,127.7,127.6,127.3,127.2,126.6,126.0,126.0,125.8,120.4,119.3,98.6(C-1a),97.2(C-1b),95.2(C-1c),82.4,80.0,79.9,78.7,77.8,75.8,75.3,75.1,73.8,73.2,71.8,69.1,68.9,63.2,63.0.HRMS(ESI)m/z calcd for C100H92F3NO19Na[M+Na]+1690.6108,found 1690.6102.
实施例6
五糖受体39的组装,路线为图5。
具体试验操作和步骤:
化合物33:根据反应条件4.1,糖基供体8(1.32g,1.7mmol)和糖基受体16(1.33g,2.0mmol)反应得到化合物33(1.17mg,55%)。[α]22
D=71.9(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.09–8.03(m,2H,Ar),7.85–7.51(m,10H,Ar),7.47–7.05(m,32H,Ar),5.46(d,J=3.6Hz,1H,H-1a),5.37(d,J=1.7Hz,1H,H-1b),5.03–4.86(m,3H,ArCH),4.81–4.60(m,9H,ArCH),4.59(m,1H,H-6a),4.53–4.43(m,3H,H-6a’,2×ArCH),4.35–4.24(m,3H,H-4b,H-5b,H-2b),4.19(ddd,J=10.0,5.5,1.9Hz,1H,H-5a),4.12(dd,J=9.2,9.2Hz,1H,H-3a),4.02(dd,J=7.0,4.7Hz,1H,H-6b),3.93(dd,J=8.6,2.6Hz,1H,H-3b),3.85(dd,J=10.5,4.7Hz,1H,H-7b),3.77(dd,J=10.4,7Hz,1H,H-7b’),3.63–3.53(m,2H,H-2a,H-4a),2.65–2.43(m,2H,SCH2CH3),1.20(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ166.3,138.9,138.5,138.4,137.7,137.7,136.1,136.1,133.3,133.3,133.1,132.9,132.9,129.8,129.8,128.5,128.5,128.4,128.2,128.2,128.2,128.0,128.0,127.9,127.9,127.8,127.6,127.6,127.5,127.3,127.3,127.2,126.5,126.0,126.0,125.9,125.9,125.8,125.6,125.6,97.1(C-1a),83.8(C-1b),81.8,81.4,79.8,79.1,77.8,75.8,75.4,75.1,74.6,74.6,73.3,72.9,72.8,72.6,71.5,71.5,69.6,63.9,25.4,14.9.HRMS(ESI)m/z calcd for C79H78O12SNa[M+Na]+1273.5106,found 1273.5109.
化合物34:根据反应条件4.2,化合物36(180mg,0.14mmol)脱去酯基得到化合物34(161mg,当量)。[α]22
D=101.2(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.81–7.56(m,8H,Ar),7.46–7.05(m,31H,Ar),5.40(d,J=3.6Hz,1H,H-1a),5.33(d,J=1.7Hz,1H,H-1b),4.98–4.86(m,3H,ArCH),4.80–4.55(m,9H,ArCH),4.48(t-like,J=11.0Hz,2H,ArCH),
4.30–4.21(m,2H,H-4b,H-5b),4.18(dd,J=1.7,2.2Hz,1H,H-2b),4.07(dd,J=9.3,9.3Hz,1H,H-3a),4.01(dd,J=6.9,4.7Hz,1H,H-6b),3.92(dd,J=8.3,2.2Hz,1H,H-3b),3.89–3.71(m,5H,H-5a,H-6a,H-6a’,H-7b,H-7b’),3.59–3.48(m,2H,H-2,H-4),2.72–2.51(m,2H,SCH2CH3),1.67(dd,J=7.4,5.3Hz,1H,OH),1.26(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ138.9,138.5,138.4,138.1,137.8,136.3,136.1,133.3,133.3,132.9,128.5,128.2,128.2,128.1,127.9,127.9,127.9,127.9,127.8,127.6,127.5,127.3,127.3,126.3,126.1,125.9,125.9,125.7,125.6,125.6,97.4(C-1a),83.7(C-1b),81.7,81.4,79.8,79.1,75.6,75.3,75.1,74.9,74.6,73.3,73.0,72.9,72.5,71.6,71.5,71.5,62.0,25.5,15.1.HRMS(ESI)m/z calcd for C72H74O11SNa[M+Na]+1169.4844,found 1169.4828.
化合物35:根据反应条件4.1,糖基供体8(350mg,0.46mmol)和糖基受体37(400mg,0.35mmol)反应得到化合物35(380mg,63%)。[α]22
D=48.8(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.01–7.94(m,2H,Ar),7.83–7.58(m,13H,Ar),7.54–7.04(m,46H,Ar),5.42(d,J=3.6Hz,1H,H-1b),5.36(d,J=1.7Hz,1H,H-1c),5.03(d-like,J=11.1Hz,1H,ArCH),5.00(d,J=3.5Hz,1H,H-1a),4.98–4.86(m,4H,ArCH),4.77–4.58(m,12H,ArCH),4.57–4.53(m,1H,H-6a),4.52–4.37(m,3H,H-6a’,2×ArCH),4.35–4.26(m,3H,H-2c,H-5c,ArCH),4.25–4.17(m,2H,H-3a,H-4c),4.14(ddd,J=10.3,4.8,2.0Hz,1H,H-5a),4.06(dd,J=9.3,9.3Hz,1H,H-3b),4.03–3.95(m,2H,H-5b,H-6c),3.91(dd,J=8.8,2.3Hz,1H,H-3c),3.90–3.79(m,2H,H-6b,H-7c),3.81–3.69(m,2H,H-6b’,H-7c’),3.66–3.59(m,2H,H-2a,H-4a),3.56(dd,J=9.3,9.3Hz,1H,H-4b),3.36(dd,J=9.3,3.6Hz,1H,H-2b),2.70–2.50(m,2H,SCH2CH3),1.19(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ166.2,138.9,138.6,138.4,138.4,138.2,137.9,137.8,136.4,136.2,136.1,133.4,133.3,133.3,133.0,133.0,132.9,132.9,130.0,129.6,128.4,128.4,128.4,128.2,128.2,128.1,128.1,128.0,127.9,127.9,127.9,127.8,127.8,127.7,127.6,127.6,127.5,127.3,127.2,127.2,126.7,126.3,126.2,126.0,126.0,125.9,125.8,125.7,125.6,125.6,96.7(C-1a,C-1b),83.9(C-1c),81.9,81.8,80.4,79.8,79.3,75.8,75.6,75.3,75.1,74.6,73.4,73.2,72.8,72.7,72.5,72.5,71.6,71.2,71.1,68.9,66.0,63.6,25.5,15.0.HRMS(ESI)m/z calcd for C110H108O17SNa[M+Na]+1755.7199,found 1755.7195.
化合物36:根据反应条件4.2,化合物35(58mg,0.033mmol)脱去酯基得到化合物36(52.6mg,98%)。[α]22
D=73.2(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ7.82–7.66(m,13H,Ar),7.48–7.02(m,43H,Ar),5.41(d,J=3.6Hz,1H,H-1b),5.36(d,J=1.7Hz,1H,H-1c),5.13(d-like,J=11.2Hz,1H,ArCH),5.00(d,J=3.4Hz,1H,H-1a),5.00–4.89(m,5H,ArCH),4.76–4.44(m,13H,ArCH),4.36(d-like,J=12.0Hz,1H,ArCH),4.32–4.20(m,3H,
H-1c,H-4c,H-5c),4.12(dd,J=8.5,8.5Hz,1H,H-3a),4.08(dd,J=8.7,8.7Hz,1H,H-3b),4.03–3.96(m,2H,H-5b,H-6c),3.95–3.87(m,2H,H-6b,H-3c),3.84(dd,J=10.5,4.6Hz,1H,H-7c),3.81–3.64(m,6H,H-5a,H-6a,H-6a’,H-4b,H-6b’,H-7c’),3.60–3.53(m,2H,H-2a,H-4a),3.44(dd,J=9.6,3.6Hz,1H,H-2b),2.69-2.53(m,2H,SCH2CH3),1.22(t,J=7.4Hz,3H,SCH2CH3).13C NMR(101MHz,CDCl3)δ138.9,138.6,138.5,138.4,138.3,138.3,137.8,136.4,136.4,136.1,133.3,133.3,133.3,132.9,132.9,132.9,128.5,128.4,128.3,128.2,128.1,128.1,128.0,128.0,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.6,127.5,127.3,127.2,127.2,126.5,126.4,126.1,126.0,125.9,125.9,125.7,125.7,125.6,125.6,97.2(C-1a),97.0(C-1b),83.8(C-1c),81.8,81.7,80.3,79.9,79.2,77.7,75.6,75.6,75.3,75.1,75.0,74.6,73.8,73.2,72.8,72.5,72.4,71.5,71.4,71.3,70.9,66.2,62.0,25.5,15.0.HRMS(ESI)m/z calcd for C103H104O16SNa[M+Na]+1651.6937,found 1651.6931.
化合物37:根据反应条件4.1,糖基供体8(163mg,0.21mmol)和糖基受体36(257mg,0.16mmol)反应得到37(230mg,65%)。[α]22
D=133.8(c1.0,CH3Cl).1H NMR(400MHz,Chloroform-d)δ8.02–7.92(m,2H,Ar),7.85–7.01(m,76H,Ar),5.42(d,J=3.6Hz,1H,H-1c),5.34(d,J=1.7Hz,1H,H-1d),5.13(d,J=3.6Hz,1H,H-1a),5.13–5.08(m,2H,ArCH),5.01–4.87(m,8H,H-1b,7×ArCH),4.81–4.52(m,15H,ArCH),4.52–4.37(m,3H,H-6a,H-6a’,ArCH),4.35–4.17(m,4H,H-2d,H-4d,H-5d,ArCH),4.16–4.04(m,3H,H-3a,H-3b,H-3c),4.04–3.90(m,5H,H-5a,H-5c,H-6c,H-3d,H-6d),3.88–3.80(m,4H,H-4a,H-5c,H-6b,H-7d),3.81–3.66(m,4H,H-4b,H-6b’,H-6c’,H-7d’),3.64(dd,J=10.1,8.9Hz,1H,H-4c),3.59(dd,J=9.6,3.5Hz,1H,H-2a),3.45(dd,J=10.0,3.5Hz,1H,H-2c),3.43(dd,J=10.0,3.5Hz,1H,H-2b),2.73–2.39(m,2H,SCH2CH3),1.17(t,J=7.4Hz,3H,SCH2CH3);13C NMR(101MHz,CDCl3)δ166.2,138.9,138.6,138.5,138.4,138.3,137.9,137.8,136.5,136.1,136.0,133.3,133.3,133.3,133.0,132.9,132.9,132.9,129.9,129.6,128.4,128.4,128.4,128.4,128.2,128.2,128.1,128.1,128.0,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.5,127.5,127.5,127.3,127.2,127.1,126.8,126.5,126.3,126.2,126.2,126.0,126.0,125.9,125.9,125.9,125.8,125.6,125.6,97.2(C-1c),97.1(C-1a,C-1b),83.9(C-1d),81.8,80.5,80.3,80.0,79.2,75.8,75.6,75.3,75.1,74.6,73.7,73.2,72.8,72.5,72.4,72.1,71.5,71.4,70.9,68.9,66.1,65.4,63.3,29.7,25.5,15.0.HRMS(ESI)m/z calcd for C141H138O22SNa[M+Na]+2237.9292,found 2237.9287.
化合物38:根据反应条件4.1,糖基供体37(150mg,0.068mmol)和糖基受体21(113mg,0.136mmol),NIS(30.6mg,0.136mmol),TfOH(0.88uL,0.01mmol),
反应温度为-20℃–0℃,反应时间为6h,得到化合物38(148mg,73%)。[α]22
D=62.5(c1.0,CH3Cl).1H NMR(700MHz,Chloroform-d)δ8.07–7.97(m,2H,Ar),7.84–6.84(m,101H,Ar),5.68(d,J=3.5Hz,1H,H-1c),5.17(d,J=3.5Hz,1H,H-1a),5.16–5.02(m,4H,ArCH),4.99–4.95(m,3H,H-1b,2×ArCH),4.94–4.85(m,6H),4.81(m,2H,H-1d,ArCH),4.77–4.64(m,9H),4.63–4.58(m,6H,H-1e),4.56–4.46(m,5H),4.46–4.38(m,8H),4.33(dd,J=12.1,3.1Hz,1H),4.28–4.15(m,5H),4.08–3.99(m,3H),3.98–3.92(m,2H),3.92–3.86(m,3H),3.85–3.78(m,3H),3.77–3.67(m,4H),3.66–3.57(m,4H),3.50(dd,J=9.6,3.4Hz,1H,H-2b),3.49–3.44(m,2H,H-2c,H-6b),3.41(dd,J=9.4,3.5Hz,1H,H-2a),3.29–3.06(m,3H,OCH2,NCH2),1.86(s,3H,OAc),1.52–1.36(m,4H,CH2CH2),1.22–1.04(m,2H,CH2);13C NMR(176MHz,CDCl3)δ170.6,166.1,138.9,138.8,138.8,138.7,138.6,138.5,138.3,138.0,137.7,136.7,136.5,136.0,136.0,133.3,133.3,133.2,133.0,133.0,132.9,132.8,132.8,130.0,129.6,128.6,128.5,128.5,128.4,128.4,128.3,128.3,128.2,128.2,128.2,128.1,128.1,128.1,128.0,128.0,127.9,127.9,127.9,127.9,127.8,127.8,127.7,127.7,127.6,127.6,127.6,127.6,127.5,127.5,127.5,127.4,127.4,127.4,127.3,127.3,127.1,127.0,126.9,126.6,126.3,126.2,126.1,126.0,126.0,126.0,125.9,125.9,125.8,125.7,125.6,125.6,125.6,125.5,125.5,97.3(C-1a,C-1e),97.2(C-1b),96.4(C-1d),95.7(C-1c),84.6,82.1,81.7,81.7,80.7,80.6,80.4,79.4,77.8,76.5,75.8,75.5,75.4,75.1,74.8,74.7,74.6,73.2,72.6,72.5,71.9,71.9,71.8,71.7,71.5,70.9,70.9,68.8,67.8,67.1,66.1,65.4,64.4,63.3,50.5,50.2,47.1,46.1,29.2,28.0,27.5,23.4,20.9.HRMS(ESI)m/z calcd for C187H187NO31Na2[M+2Na]2+3030.2977,found 1515.1508.
化合物39:将化合物38(126mg)溶解到DCM/MeOH(1/1,v/v,2.6mL)中,冷却至0℃,加入AcCl(0.26mL)。反应液在室温下反应24h。反应结束后反应液用DCM稀释,用NaHCO3洗涤,有机相用无水Na2SO4干燥,层析柱分离纯化得到化合物39(101mg,82%)。[α]22
D=39.5(c0.9,CH3Cl).1H NMR(700MHz,Chloroform-d)δ8.01(d,J=7.8Hz,2H),7.84–6.78(m,101H),5.72(d,J=3.4Hz,1H,H-1c),5.18(d,J=3.4Hz,2H,H-1a,ArCH),5.18–5.12(m,2H),5.09–5.02(m,3H,H-1b,2×ArCH),5.00–4.87(m,7H),4.87–4.78(m,3H,H-1d,2×ArCH),4.76–4.54(m,15H,H-1e),4.51–4.40(m,11H),4.39–4.27(m,3H),4.27–4.15(m,3H),4.12–4.02(m,3H),3.98–3.72(m,14H),3.71–3.52(m,8H),3.48–3.41(m,2H),3.29–3.06(m,3H,OCH2,NCH2),1.52–1.35(m,4H,CH2CH2),1.23–1.05(m,2H,CH2);13C NMR(176MHz,CDCl3)δ192.4,192.3,166.2,156.7,156.2,139.0,138.7,138.7,138.7,138.6,138.6,138.5,138.1,138.0,137.9,137.7,136.9,136.7,136.5,136.5,136.1,136.0,134.6,134.5,133.4,133.3,133.3,133.2,133.1,133.0,132.9,132.9,132.8,132.7,130.0,129.8,129.7,129.6,129.1,129.0,
128.6,128.6,128.5,128.5,128.4,128.4,128.4,128.3,128.3,128.3,128.3,128.2,128.2,128.2,128.1,128.0,128.0,127.9,127.9,127.8,127.8,127.8,127.7,127.7,127.6,127.6,127.6,127.6,127.5,127.5,127.5,127.4,127.4,127.3,127.3,127.2,127.2,127.2,127.1,127.1,127.0,127.0,126.6,126.4,126.3,126.1,126.1,126.0,126.0,125.9,125.9,125.9,125.8,125.8,125.7,125.7,125.6,125.5,122.8,97.4(C-1a,C-1e),97.2(C-1b),96.6(C-1d),95.7(C-1c),84.7,82.0,81.7,81.7,80.7,80.5,80.4,79.4,78.7,78.1,77.7,77.4,77.3,75.9,75.5,75.5,75.3,75.2,74.9,74.8,74.8,74.7,73.2,72.7,72.5,72.1,71.9,71.9,71.8,71.8,71.5,71.0,70.9,68.9,67.7,67.2,66.1,65.4,63.2,61.8,50.5,50.2,47.1,46.2,29.3,28.0,27.5,23.4.HRMS(ESI)m/z calcd for C189H189NO32Na[M+Na]+2965.2979,found 2965.2993.
实施例7
目标八糖41的合成,路线为图6。
具体试验操作和步骤:
化合物40:三糖供体32(38.0mg,0.015mmol)和五糖受体39(29.4mg,0.01mmol)与甲苯共蒸三次。加入干燥的DCM(1.0mL),加入活化的4
分子筛,溶液在室温下搅拌30min后冷却至0℃,加入TMSOTf(0.05MinDCM,30uL,1.5umol),在0℃下搅拌6h。反应结束后用吡啶终止反应。反应液过滤,加入DCM稀释后用饱和的NaHCO
3洗涤。无水Na
2SO
4干燥浓缩。柱层析纯化分离得到化合物40(24.8mg,56%)。[α]
22
D=62.8(c1.0,CH
3Cl).
1H NMR(700MHz,Chloroform-d)δ8.06–7.86(m,8H),7.83–6.88(m,145H),5.60(d,J=3.4Hz,1H,H-1c),5.58(m,2H,H-1h,H-2f),5.10(m,2H),5.07(d,J=3.8Hz,2H,H-1a,H-1g),5.07–4.98(m,6H),4.98–4.87(m,10H,H-1b),4.87–4.81(m,2H),4.80–4.72(m,4H,H-1d),4.72–4.62(m,11H),4.62–4.48(m,18H,H-1e),4.48–4.26(m,26H,H-1f),4.25–4.17(m,8H),4.16–4.10(m,3H),4.09–4.01(m,3H),4.01–3.92(m,5H),3.93–3.84(m,5H),3.82–3.64(m,7H),3.63–3.45(m,12H),3.45–3.29(m,6H),3.25–2.93(m,3H,OCH
2,NCH
2),1.41–1.31(m,2H,CH
2),1.24–1.14(m,2H,CH
2),1.09–0.88(m,2H,CH
2).
13C NMR(176MHz,CDCl
3)δ166.1,166.0,165.0,139.4,139.0,138.9,138.9,138.8,138.7,138.7,138.6,138.4,138.2,138.1,138.1,138.0,138.0,137.7,137.6,137.4,136.9,136.5,136.4,136.1,136.1,136.0,133.4,133.3,133.3,133.3,133.1,133.0,133.0,132.9,132.9,132.9,132.8,132.7,130.3,130.2,130.2,130.1,129.9,129.8,129.7,129.7,129.6,129.0,129.0,128.7,128.6,128.6,128.5,128.5,128.4,128.4,128.4,128.4,128.3,128.3,128.3,128.3,128.3,128.2,128.2,128.1,128.1,128.0,128.0,128.0,127.9,127.9,127.9,127.9,127.8,127.7,127.7,127.7,127.6,127.6,127.6,127.6,127.5,
127.5,127.4,127.4,127.4,127.3,127.3,127.2,127.2,127.1,127.1,126.9,126.9,126.8,126.7,126.6,126.4,126.4,126.4,126.1,126.1,126.1,126.0,126.0,125.9,125.9,125.8,125.8,125.7,125.7,125.6,102.1(C-1f),98.4(C-1a,C-1g),97.7(C-1b),97.2(C-1e),97.1(C-1h),96.1(C-1c,C-1d),84.3,82.5,82.2,81.9,81.7,80.8,80.5,80.4,80.3,80.2,79.3,79.1,78.5,78.4,77.9,76.6,76.1,75.8,75.8,75.6,75.6,75.1,75.1,74.9,74.6,74.3,74.2,73.6,73.5,73.3,73.3,73.1,72.7,72.6,72.5,71.9,71.7,71.7,71.6,71.4,70.8,70.2,69.0,68.8,68.6,67.8,67.6,67.1,65.6,63.4,63.3,63.2,50.5,50.1,47.1,46.2,44.8,32.0,29.7,29.5,29.4,29.1,27.9,27.5,23.2,22.7.TheJ
C-Hcouplingconstantswere176.4Hz(c),174.4Hz(h),170.0Hz(a),170.4Hz(g),170.4Hz(b),168.8Hz(d),170.4Hz(e),163.6Hz(f).HRMS(ESI)m/z calcd for C
279H
273NO
49Na
2[M+2Na]
2+4466.8686,found 2233.4497.
化合物41:化合物41为式I结构中Rn都为H。将化合物40(10mg,2.3ummol)溶解在MeOH/THF(1:1,v/v,1mL)中,加入15%NaOH(100uL)搅拌1h,加入MeONa(50mg)。溶液在室温下搅拌12h,反应液加入AmberliteIR120H+树脂中和至PH为7。过滤浓缩,柱层析纯化分离,得到半脱保护产物。半脱保护产率溶解于DCM/t-Butanol/H2O(3:6:1,v/v/v,2mL)中,加入Pd/C。混合物在1个大气压的H2压力下反应后三天,过滤浓缩。用C18反相柱纯化,得到全脱保护产物41(2.1mg,63%)。1H NMR(700MHz,D2O)δ5.37(d,J=3.7Hz,1H,H-1c),5.35(d,J=3.9Hz,1H,H-1f),5.02(d,J=3.7Hz,1H,H-1a),4.99(d,J=3.7Hz,1H,H-1g),4.96(s,1H,H-1d),4.95(d,J=3.9Hz,1H,H-1h),4.79(s,1H,H-1e),4.52(d,J=7.8Hz,1H,H-1f),4.24(d,J=2.9Hz,1H),4.23–4.16(m,3H),4.13–4.08(m,2H),4.08–4.01(m,4H),3.99(dt,J=10.2,3.2Hz,1H),3.95–3.90(m,2H),3.89–3.67(m,25H),3.67–3.59(m,4H),3.59–3.52(m,7H),3.51–3.41(m,3H),3.02(t,J=7.6Hz,2H),1.74–1.62(m,4H),1.52–1.42(m,2H).13C NMR(176MHz,D2O)δ103.6,100.1,99.5,99.1,98.7,97.8,97.7,97.4,80.0,76.9,76.8,76.4,75.2,74.0,73.4,73.1,73.0,72.6,72.1,72.1,72.0,71.8,71.7,71.6,71.5,71.5,70.9,70.8,70.7,70.7,70.5,70.4,70.3,69.7,69.5,69.4,69.2,69.2,68.0,67.7,67.6,65.2,62.5,61.8,60.5,60.2,60.0,59.8,39.4,28.0,26.6,22.5.HRMS(ESI)m/z calcd for C55H97NO43Na[M+H]+1460.5507,found 1460.547。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。