CN107698629A - 二硫键桥连去垢剂及其在膜蛋白研究中的应用 - Google Patents
二硫键桥连去垢剂及其在膜蛋白研究中的应用 Download PDFInfo
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
本发明提供了一种二硫键桥连去垢剂及其在膜蛋白研究中的应用。所述的去垢剂分子,其特征在于,其结构式为:
Description
技术领域
本发明涉及表面活性剂领域。
背景技术
去垢剂是同时具有亲水极性基团和疏水非极性基团的双亲性分子。它能够提取纯化质膜上的膜蛋白,并在溶液中维持其结构和功能的稳定性,是膜蛋白研究中必不可少的工具。膜蛋白研究中去垢剂使用剂量较大,而且不同的膜蛋白或者同一膜蛋白的不同处理阶段以及不同的研究方法对去垢剂的性能要求不一样,因此该领域对于去垢剂的产业化和多元化皆有很高要求。然而,目前可供选择的商业化的传统去垢剂种类有限,无法完全满足多种膜蛋白体系及其不同结构和功能研究手段的需求。另外,近年发展的若干新型去垢剂,如高分子去垢剂Amphipol和短肽去垢剂等,由于合成难度问题无法达到商业化试剂的生产规模要求,未能获得大范围的推广使用。
发明内容
本发明的目的是开发用于膜蛋白研究的去垢剂分子。该类去垢剂分子能够分离纯化并稳定膜蛋白的生理结构和功能,进而可应用于下游的膜蛋白结构和活性研究。
为了达到上述目的,本发明采用了如下技术方案:
一种二硫键桥连的去垢剂分子,其特征在于,其结构式为:
其中,A为未取代的或取代的单糖和取代的二糖,以及各种形式的铵盐中的一种,B为氢或者含有或不含有杂原子的脂肪族取代基;X是含有或不含有杂原子的脂肪族线性亚基。
优选地,所述的A为下述结构中的一种:
其中,x为0-3之间的整数,y为1-6之间的整数。
优选地,所述的含有或不含有杂原子的脂肪族取代基为下述结构中的一种:
其中m为0-20之间的整数。
优选地,所述的X为O(CH2)n,S(CH2)n,NH(CH2)n或NHC(O)(CH2)n,其中n为0-20之间的整数。
优选地,所述的A为未取代的或取代的单糖和取代的二糖,B为不含有杂原子的直链的脂肪族取代基,所述的X为O(CH2)n,S(CH2)n,NH(CH2)n或NHC(O)(CH2)n,其中n为0-20之间的整数。
优选地,所述的A为未取代的二糖,B为直链的脂肪族取代基,X是O(CH2)n,其中n为0-20之间的整数。
优选地,所述的二硫键桥连的去垢剂分子,其特征在于,其结构式为:
本发明还提供了上述的二硫键桥连的去垢剂分子的制备方法,其特征在于,包括:
步骤1:beta-D-麦芽糖八乙酸酯加入混有分子筛的溶剂中溶解,然后加入第一溴醇化合物;将上述混合物在氮气环境中搅拌,加入三氟化硼乙醚,搅拌过夜;反应结束后,过滤除去分子筛,分出有机相,分离得到第一中间体;
步骤2:将第一中间体溶于溶剂中,加入四丁基溴化铵和硫代苯磺酸钠进行反应,后处理得到第二中间体;
步骤3:将第二中间体溶于溶剂中,加入三乙胺,搅拌,逐滴加入硫醇化合物溶液,搅拌,后处理得到第三中间体;
步骤4:将第三中间体溶于溶剂中,加入甲醇钠的甲醇溶液,搅拌,后处理得到二硫键桥连的去垢剂分子。
本发明还提供了上述的二硫键桥连的去垢剂分子的衍生物,其特征在于,为上述的二硫键桥连的去垢剂分子的对映异构体、非对映异构体、几何异构体、互变异构体、旋转异构体、阻转异构体、消旋体、代谢产物、盐类、水合物或高聚物。
本发明还提供了上述的二硫键桥连的去垢剂分子或其衍生物在制备膜蛋白去垢剂中的应用。
本发明还提供了上述的二硫键桥连的去垢剂分子或其衍生物在膜蛋白分离纯化、离体表达以及结构功能研究的至少一种中的应用。
一种去垢剂,其特征在于,其含有上述的二硫键桥连的去垢剂分子及其衍生物。
本发明在膜蛋白研究中常用的传统去垢剂的结构基础上,引入二硫键来桥连其疏水性非极性部分,利用二硫键独特的二面角结构,改变去垢剂分子的拓扑结构,进而影响其的胶束性质。本发明在已知去垢剂β-十二烷基麦芽糖苷(DDM)的基础上,在烷基链的不同位置插入二硫键,得到了一系列二硫键桥连去垢剂。评价实验发现,DID-1-L极大地稳定了腺苷受体,在相关的蛋白纯化和稳定性实验中,其稳定蛋白的作用甚至优于DDM(图3b)。同时,DLS结果皆显示二硫键去垢剂形成更小的胶束(图2),NMR实验进一步验证了其在膜蛋白溶液NMR领域的应用潜力(图4)。
受到以上结果的鼓舞,本发明拓展了小分子去垢剂的设计思路,从已知传统去垢剂出发,在适当位置引入二硫键。以不同的已知化合物为母体,通过改变二硫键的位置和数目,设计合成了多个系列的化合物。已知母体化合物包括,但不限于DDM、LMNG、Fos-choline等。所列小分子配体符合通式以及相关联的对映异构体、非对映异构体、几何异构体、互变异构体、旋转异构体、阻转异构体、消旋体、代谢产物等,以及相应的盐类、水合物、高聚物等不同形式。
与现有技术相比,本发明的有益效果是:
本发明开发了用于膜蛋白研究的一类新型小分子去垢剂。本发明通过在小分子的相应位置引入二硫键,改变去垢剂分子的拓扑构象,使其相对于原型去垢剂具有优秀的溶膜能力,更好的蛋白稳定作用,以及更小的胶束体积。同时,本发明明显提高了膜蛋白核磁信号的分辨率。此类小分子去垢剂可以作为工具分子应用于膜蛋白的功能和结构研究。
附图说明
图1为除去垢剂DID-1-L,DID-3-S和DID-3-L外其他二硫键桥连去垢剂的合成反应通式。
图2二硫键桥连去垢剂与传统去垢剂DDM和LDAO的DLS结果。
图3(a)二硫键桥连去垢剂和DDM纯化的腺苷受体2A亚型蛋白的分析凝胶排阻层析色谱图。(b)腺苷受体2A亚型蛋白分别溶解在DID-1-L和DDM环境中的荧光热稳定实验结果。
图4细菌外膜蛋白OmpX分别溶解在DID-7(a)和DDM(b)中的2D[15N,1H]-TROSY相关NMR谱图效果对比。
具体实施方式
下面结合具体实施例,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,在阅读了本发明讲授的内容之后,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。
实施例1:二硫键桥连的去垢剂分子及其合成
1、二硫键桥连去垢剂,其结构式分别为(I)-(XI)。
2、除去垢剂DID-1-L,DID-3-S和DID-3-L外其他二硫键桥连去垢剂合成方法如图1所示,试剂和反应条件:i)beta-D-麦芽糖八乙酸酯,三氟化硼乙醚,二氯甲烷,0℃到室温;ii)硫代苯磺酸钠,四丁基溴化铵,乙腈,70℃;iii)三乙胺,二氯甲烷,0℃到室温;iv)甲醇钠,甲醇,室温。
具体步骤如下:
(i)将10.9g(16mmol)beta-D-麦芽糖八乙酸酯(韶远,货号SY059282)加入混有2g分子筛(泰坦,货号G78421A)的30ml干燥二氯甲烷中溶解,然后加入10mmol溴醇化合物1。上述混合物在氮气环境中0℃搅拌10分钟后,慢慢加入80mmol三氟化硼乙醚(阿拉丁,货号B104431),于室温下继续在氮气环境中搅拌过夜。反应结束后加入20ml饱和碳酸氢钠水溶液淬灭,过滤除去分子筛,分出有机相,水相用二氯甲烷(每次20ml)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体2a-2h。
其中,结构式分别为2a-2h的中间体对应的溴醇化合物1a-1h均为市售产品,见下表:
中间体2a-2h结构表征,具体如下:
2a:白色固体,分离产率71%。1H NMR(800MHz,CDCl3)δ5.39(d,J=4.1Hz,1H),5.33(dd,J=10.5,9.5Hz,1H),5.24(t,J=9.2Hz,1H),5.05-5.01(m,1H),4.84-4.80(m,2H),4.57(d,J=7.9Hz,1H),4.47(dd,J=12.1,2.8Hz,1H),4.21(ddd,J=20.0,12.3,4.2Hz,2H),4.12-4.08(m,1H),4.02(dd,J=12.4,2.3Hz,1H),4.00-3.96(m,1H),3.93(ddd,J=10.3,4.0,2.3Hz,1H),3.79(ddd,J=11.4,7.5,6.1Hz,1H),3.67(ddd,J=9.7,4.5,2.8Hz,1H),3.46-3.39(m,2H),2.12(s,3H),2.08(s,3H),2.02(d,J=1.5Hz,6H),2.00(s,3H),1.98(s,3H),1.98(s,3H).13C NMR(201MHz,CDCl3)δ170.61,170.51,170.27,170.04,169.78,169.50,100.57,95.64,75.25,72.70,72.36,72.00,70.09,69.87,69.41,68.62,68.12,62.76,61.60,29.93,20.99,20.93,20.80,20.78,20.70,20.66.高分辨质谱C28H39O18Br[M+Na]+计算值:765.1217;实测值:765.1212。
2b:白色固体,分离产率50%。1H NMR(500MHz,CDCl3)δ5.40(d,J=4.0Hz,1H),5.35(t,J=10.0Hz,1H),5.25(t,J=9.2Hz,1H),5.04(t,J=9.9Hz,1H),4.83(ddd,J=17.5,10.0,5.9Hz,2H),4.52(d,J=7.9Hz,1H),4.48(dd,J=12.2,2.8Hz,1H),4.23(td,J=12.0,11.5,4.2Hz,2H),4.06-3.90(m,4H),3.70-3.64(m,2H),3.44(ddd,J=8.0,5.9,4.0Hz,2H),2.13(s,3H),2.09(s,3H),2.05-2.00(m,9H),1.99(s,6H),1.91-1.81(m,2H).13C NMR(126MHz,CDCl3)δ170.68,170.61,170.33,170.09,169.84,169.56,100.63,95.61,75.41,72.71,72.25,72.21,70.09,69.43,68.60,68.09,67.45,62.86,61.59,32.37,30.15,21.04,21.00,20.83,20.81,20.75,20.73,20.71.高分辨质谱C29H41O18Br[M+Na]+计算值:779.1374;实测值:779.1370。
2c:白色固体,分离产率31%。1HNMR(500MHz,CDCl3)δ5.38(d,J=3.3Hz,1H),5.33(td,J=9.9,2.2Hz,1H),5.22(td,J=9.2,2.0Hz,1H),5.02(td,J=9.9,2.2Hz,1H),4.87-4.75(m,2H),4.49(d,J=8.2Hz,1H),4.46(d,J=12.2Hz,2H),4.21(t,J=14.0Hz,1H),4.04-3.96(m,2H),3.96-3.83(m,2H),3.69-3.61(m,1H),3.53-3.46(m,1H),3.38(t,J=7.0Hz,2H),2.12(d,J=2.1Hz,3H),2.07(d,J=2.1Hz,3H),2.03-1.96(m,15H),1.88(m,2H),1.70(q,J=7.0Hz,2H).13C NMR(126MHz,CDCl3)δ170.63,170.57,170.34,170.06,169.72,169.52,100.30,95.59,75.46,72.74,72.20,70.07,69.40,69.01,68.56,68.06,62.85,61.56,33.37,29.37,28.07,21.01,20.96,20.79,20.77,20.72,20.69,20.67.高分辨质谱C30H43O18Br[M+Na]+计算值:793.1530;实测值:793.1474。
2d:白色固体,分离产率42%。1HNMR(500MHz,CDCl3)δ5.39(d,J=3.9Hz,1H),5.33(t,J=10.0Hz,1H),5.22(t,J=9.2Hz,1H),5.02(t,J=9.9Hz,1H),4.83(dd,J=10.5,4.0Hz,1H),4.78(dd,J=9.5,7.9Hz,1H),4.49(d,J=7.9Hz,1H),4.45(dd,J=12.1,2.7Hz,1H),4.21(td,J=12.5,4.0Hz,2H),4.04-3.95(m,2H),3.96-3.91(m,1H),3.84(dt,J=9.7,6.1Hz,1H),3.64(ddt,J=9.4,5.0,2.5Hz,1H),3.46(dt,J=9.8,6.5Hz,1H),3.36(td,J=6.8,2.2Hz,2H),2.12(s,2H),2.07(s,2H),2.03-1.96(m,15H),1.83(m,2H),1.61-1.51(m,2H),1.45(m,2H).13C NMR(126MHz,CDCl3)δ170.61,170.55,170.34,170.04,169.70,169.50,100.33,95.57,75.49,72.75,72.23,72.15,70.05,69.79,69.39,68.54,68.06,62.88,61.55,33.74,32.39,28.62,24.66,20.96,20.78,20.71,20.68,20.66.高分辨质谱C31H45O18Br[M+Na]+计算值:807.1687;实测值:807.1681。
2e:白色固体,分离产率30%。1HNMR(800MHz,CDCl3)δ5.38(d,J=4.1Hz,1H),5.32(dd,J=10.5,9.5Hz,1H),5.23-5.20(m,1H),5.01(dd,J=10.3,9.5Hz,1H),4.82(dd,J=10.5,4.0Hz,1H),4.77(dd,J=9.5,7.9Hz,1H),4.48(d,J=7.9Hz,1H),4.44(dd,J=12.1,2.8Hz,1H),4.21(ddd,J=13.9,12.3,4.2Hz,2H),4.01(dd,J=12.4,2.3Hz,1H),3.96(dd,J=9.6,8.8Hz,1H),3.93(ddd,J=10.3,4.1,2.4Hz,1H),3.81(dt,J=9.7,6.3Hz,1H),3.64(ddd,J=9.6,4.4,2.8Hz,1H),3.45(dt,J=9.7,6.7Hz,1H),3.36(t,J=6.8Hz,2H),2.11(s,3H),2.07(s,3H),2.01(s,3H),1.99(s,3H),1.98(s,3H),1.97(d,J=1.0Hz,6H),1.81(dt,J=14.3,6.9Hz,2H),1.54(dddd,J=15.8,13.8,7.1,4.1Hz,2H),1.43-1.37(m,2H),1.35-1.27(m,2H).13C NMR(201MHz,CDCl3)δ170.59,170.53,170.32,170.02,169.65,169.48,100.35,95.58,75.50,72.85,72.28,72.17,70.07,69.94,69.41,68.55,68.11,62.94,61.59,33.80,32.71,29.26,27.83,25.08,20.99,20.93,20.76,20.73,20.68,20.65.高分辨质谱C32H47O18Br[M+Na]+计算值:821.1843;实测值:821.1837。
2f:白色固体,分离产率35%。1H NMR(800MHz,CDCl3)δ5.39(d,J=4.1Hz,1H),5.33(dd,J=10.5,9.5Hz,1H),5.22(dd,J=9.5,8.8Hz,1H),5.02(dd,J=10.3,9.5Hz,1H),4.83(dd,J=10.5,4.1Hz,1H),4.78(dd,J=9.5,7.9Hz,1H),4.49(d,J=7.9Hz,1H),4.45(dd,J=12.1,2.8Hz,1H),4.26-4.18(m,2H),4.02(dd,J=12.4,2.3Hz,1H),3.97(dd,J=9.6,8.8Hz,1H),3.94(ddd,J=10.2,4.0,2.3Hz,1H),3.82(dt,J=9.6,6.3Hz,1H),3.65(ddd,J=9.6,4.4,2.8Hz,1H),3.44(dt,J=9.7,6.7Hz,1H),3.37(t,J=6.8Hz,2H),2.12(s,3H),2.08(s,3H),2.02(s,3H),2.00(s,3H),1.99(s,3H),1.98(d,J=1.1Hz,6H),1.85-1.77(m,2H),1.58-1.50(m,2H),1.42-1.37(m,2H),1.36-1.20(m,4H).13C NMR(201MHz,CDCl3)δ170.59,170.53,170.32,170.02,169.65,169.48,100.35,95.58,75.50,72.85,72.28,72.17,70.07,69.94,69.41,68.55,68.11,62.94,61.59,33.80,32.71,29.26,27.83,25.08,20.99,20.93,20.76,20.73,20.68,20.65,20.65.高分辨质谱C33H49O18Br[M+Na]+计算值:835.1994;实测值835.1994。
2g:白色固体,分离产率32%。1HNMR(600MHz,CDCl3)δ5.41(d,J=4.0Hz,1H),5.35(t,J=10.1Hz,1H),5.24(t,J=9.2Hz,1H),5.04(t,J=9.9Hz,1H),4.85(dd,J=10.5,4.0Hz,1H),4.80(dd,J=9.5,7.9Hz,1H),4.50(d,J=7.9Hz,1H),4.46(dd,J=12.1,2.8Hz,1H),4.23(td,J=12.4,4.1Hz,2H),4.03(dd,J=12.4,2.3Hz,1H),3.99(t,J=9.2Hz,1H),3.95(ddd,J=10.3,3.8,2.4Hz,1H),3.83(dt,J=9.6,6.3Hz,1H),3.66(ddd,J=9.5,4.3,2.8Hz,1H),3.45(dt,J=9.6,6.7Hz,1H),3.39(t,J=6.8Hz,2H),2.13(s,3H),2.09(s,3H),2.03(s,3H),2.02(s,3H),2.01(s,3H),1.99(s,5H),1.83(dt,J=14.4,6.9Hz,2H),1.59-1.50(m,2H),1.45-1.37(m,2H),1.35-1.27(m,6H).13C NMR(151 MHz,CDCl3)δ170.67,170.62,170.40,170.10,169.72,169.55,100.41,95.60,75.57,72.82,72.32,72.16,70.26,70.10,69.45,68.58,68.10,62.99,61.60,34.12,32.87,29.45,29.22,28.81,28.19,25.83,21.07,21.01,20.84,20.80,20.76,20.73,20.72.高分辨质谱C34H51O18Br[M+Na]+计算值:849.2156;实测值:849.2152。
2h:白色固体,分离产率23%。1H NMR(500MHz,CDCl3)δ5.40(d,J=4.0Hz,1H),5.35(t,J=10.1Hz,1H),5.24(t,J=9.2Hz,1H),5.04(t,J=9.8Hz,1H),4.85(dt,J=10.5,2.8Hz,1H),4.80(t,J=8.7Hz,1H),4.50(d,J=7.9Hz,1H),4.46(dd,J=12.1,2.6Hz,1H),4.23(ddd,J=13.8,10.7,4.0Hz,2H),4.06-3.98(m,2H),3.98-3.92(m,1H),3.83(dt,J=8.7,6.4Hz,1H),3.66(dt,J=9.3,3.4Hz,1H),3.45(dt,J=9.3,6.9Hz,1H),3.42-3.35(m,3H),2.13(s,3H),2.09(s,3H),2.05-1.96(m,15H),1.83(p,J=7.1Hz,2H),1.54(q,J=6.5Hz,2H),1.40(t,J=7.4Hz,2H),1.31-1.23(m,8H).13C NMR(126MHz,CDCl3)δ170.68,170.63,170.41,170.11,169.73,169.56,100.41,95.60,75.57,72.83,72.33,72.16,70.31,70.10,69.45,68.57,68.11,63.00,61.61,34.16,32.90,29.48,29.46,29.30,28.81,28.25,25.88,21.06,21.00,20.83,20.78,20.75,20.73,20.71.高分辨质谱C35H53O18Br[M+Na]+计算值:863.2313;实测值:863.2266 and 865.2254。
(ii)3mmol中间体2a-2h溶于20ml无水乙腈中,加入100mg四丁基溴化铵(泰坦,货号:G28296B)和941mg硫代苯磺酸钠(大唐医药,货号:BL-06097)。反应液在70℃回流6小时(或者反应过夜)。反应结束后,旋蒸除掉溶剂。加入30mL乙酸乙酯和30mL水,分出有机相,水相用乙酸乙酯(每次30ml)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体3a-3h。
中间体3a-3h结构表征,具体如下:
3a:白色固体,分离产率75%。1H NMR(800MHz,CDCl3)δ7.91(dd,J=8.5,1.2Hz,2H),7.65(tt,J=7.4,1.1Hz,1H),7.57(dd,J=8.3,7.4Hz,2H),5.38(d,J=4.0Hz,1H),5.34(dd,J=10.6,9.5Hz,1H),5.21(dd,J=9.5,8.9Hz,1H),5.04(dd,J=10.3,9.5Hz,1H),4.84(dd,J=10.5,4.1Hz,1H),4.76(dd,J=9.5,7.9Hz,1H),4.48-4.45(m,2H),4.23(dd,J=12.5,3.9Hz,1H),4.18(dd,J=12.1,4.4Hz,1H),4.04-4.00(m,2H),3.97-3.92(m,2H),3.73(ddd,J=10.6,7.7,6.1Hz,1H),3.64(ddd,J=9.6,4.4,2.7Hz,1H),3.18-3.10(m,2H),2.12(s,3H),2.08(s,3H),2.03(s,3H),2.02(s,3H),2.01(s,3H),1.99(d,J=2.4Hz,6H).13CNMR(126MHz,CDCl3)δ170.64,170.54,170.25,170.06,169.77,169.52,144.67,133.99,129.54,127.07,100.55,95.66,75.25,72.66,72.40,71.94,70.10,69.41,68.63,68.12,68.07,62.74,61.60,35.59,20.99,20.95,20.81,20.80,20.72,20.69.高分辨质谱C34H44O20S2[M+Na]+计算值:859.1765;实测值:859.1755。
3b:白色固体,分离产率90%。1H NMR(500MHz,CDCl3)δ7.92(d,J=7.5Hz,2H),7.64(t,J=7.4Hz,1H),7.56(t,J=7.7Hz,2H),5.40(d,J=4.0Hz,1H),5.35(t,J=10.0Hz,1H),5.23(t,J=9.2Hz,1H),5.05(t,J=9.9Hz,1H),4.85(dd,J=10.5,4.0Hz,1H),4.77(dd,J=9.4,8.0Hz,1H),4.48-4.45(m,2H),4.24(dd,J=12.5,3.9Hz,1H),4.19(dd,J=12.2,4.4Hz,1H),4.03(dd,J=12.4,2.0Hz,1H),3.99-3.92(m,2H),3.86(dt,J=10.3,5.4Hz,1H),3.65(ddd,J=9.7,4.5,2.7Hz,1H),3.54(ddd,J=10.0,7.5,4.7Hz,1H),3.03(t,J=7.0Hz,2H),2.13(s,3H),2.09(s,3H),2.04(s,3H),2.02(s,3H),2.01-1.99(m,9H),1.97-1.85(m,2H).13C NMR(126MHz,CDCl3)δ170.71,170.70,170.62,170.34,170.13,169.84,169.58,144.74,133.85,129.48,127.08,100.29,95.64,75.41,72.70,72.28,72.12,70.10,69.42,68.62,68.09,67.49,62.81,61.59,32.69,28.90,21.04,21.00,20.84,20.81,20.76,20.73,20.72.高分辨质谱C35H46O20S2[M+Na]+计算值:873.1922;实测值:873.1916。
3c:白色固体,分离产率86%。1H NMR(800MHz,CDCl3)δ7.92(dd,J=8.4,1.2Hz,2H),7.64(t,J=7.4Hz,1H),7.56(dd,J=8.3,7.4Hz,2H),5.40(d,J=4.1Hz,1H),5.35(dd,J=10.5,9.5Hz,1H),5.25-5.20(m,1H),5.05(dd,J=10.3,9.5Hz,1H),4.85(dd,J=10.5,4.0Hz,1H),4.77(dd,J=9.5,7.9Hz,1H),4.48-4.46(m,2H),4.24(dd,J=12.5,3.9Hz,1H),4.20(dd,J=12.1,4.4Hz,1H),4.04(dd,J=12.5,2.3Hz,1H),3.99-3.96(m,1H),3.94(qd,J=5.9,5.2,1.9Hz,1H),3.81-3.78(m,1H),3.65(ddd,J=9.6,4.3,2.8Hz,1H),3.44(ddd,J=9.7,7.0,5.5Hz,1H),2.99(t,J=7.3Hz,2H),2.13(s,3H),2.09(s,3H),2.04(s,3H),2.02(s,3H),2.01-1.98(m,9H),1.73-1.66(m,2H),1.63-1.54(m,2H).13C NMR(201MHz,CDCl3)δ170.65,170.56,170.34,170.08,169.71,169.53,144.95,133.74,129.40,127.04,100.36,95.64,75.51,72.85,72.28,72.25,70.12,69.62,69.45,68.60,68.14,62.91,61.62,35.98,28.83,28.45,24.98,21.03,20.98,20.81,20.79,20.73,20.70,20.69.高分辨质谱C36H48O20S2计算值:887.2078;实测值:887.2072。
3d:白色固体,分离产率36%。1H NMR(800MHz,CDCl3)δ7.91(dd,J=8.5,1.3Hz,2H),7.63(t,J=7.4Hz,1H),7.55(dd,J=8.3,7.4Hz,2H),5.40(d,J=4.0Hz,1H),5.35(dd,J=10.5,9.5Hz,1H),5.24-5.21(m,1H),5.04(dd,J=10.3,9.5Hz,1H),4.84(dd,J=10.6,4.0Hz,1H),4.78(dd,J=9.5,7.9Hz,1H),4.48-4.45(m,2H),4.22(ddd,J=23.7,12.3,4.2Hz,2H),4.03(dd,J=12.5,2.3Hz,1H),3.97(dd,J=9.6,8.8Hz,1H),3.95(ddd,J=10.3,3.9,2.3Hz,1H),3.78(dt,J=9.7,6.2Hz,1H),3.65(ddd,J=9.7,4.4,2.8Hz,1H),3.44-3.39(m,1H),2.96(td,J=7.2,1.0Hz,2H),2.12(s,3H),2.09(s,3H),2.03(s,3H),2.01(s,3H),1.99(s,9H),1.61(p,J=7.6Hz,2H),1.53-1.44(m,2H),1.38-1.29(m,2H).13CNMR(201MHz,CDCl3)δ170.65,170.56,170.34,170.08,169.71,169.53,144.95,133.74,129.40,127.04,100.36,95.64,75.51,72.85,72.28,72.25,70.12,69.62,69.45,68.60,68.14,62.91,61.62,35.98,28.83,28.45,24.98,21.03,20.98,20.81,20.79,20.73,20.70,20.69.高分辨质谱C37H50O20S2[M+Na]+计算值:901.2235;实测值:901.2225。
3e:白色固体,分离产率86%。1H NMR(800MHz,CDCl3)δ7.92(dd,J=8.5,1.3Hz,2H),7.62(tt,J=7.5,1.3Hz,1H),7.55(dd,J=8.3,7.4Hz,2H),5.40(d,J=4.0Hz,1H),5.34(dd,J=10.5,9.5Hz,1H),5.23(t,J=9.2Hz,1H),5.04(t,J=9.9Hz,1H),4.84(dd,J=10.5,4.0Hz,1H),4.78(dd,J=9.5,7.9Hz,1H),4.48(d,J=7.9Hz,1H),4.46(dd,J=12.2,2.8Hz,1H),4.22(ddd,J=18.3,12.3,4.2Hz,2H),4.03(dd,J=12.5,2.3Hz,1H),3.98(t,J=9.2Hz,1H),3.97-3.93(m,1H),3.78(dt,J=9.6,6.4Hz,1H),3.65(ddd,J=9.7,4.4,2.9Hz,1H),3.42(dt,J=9.7,6.6Hz,1H),2.97(t,J=7.3Hz,2H),2.12(s,3H),2.09(s,3H),2.03(s,3H),2.01(s,3H),1.99(d,J=1.6Hz,9H),1.58(p,J=7.4Hz,2H),1.53-1.41(m,2H),1.31-1.20(m,4H).13C NMR(201MHz,CDCl3)δ170.65,170.58,170.35,170.08,169.69,169.53,145.00,133.71,129.38,127.04,100.38,95.62,75.52,72.87,72.31,72.23,70.12,69.91,69.45,68.59,68.15,62.95,61.62,36.04,29.21,28.68,28.24,25.29,21.03,20.97,20.80,20.77,20.72,20.70,20.69.高分辨质谱C38H52O20S2[M+Na]+计算值:915.2391;实测值:901.2440。
3f:白色固体,分离产率98%。1HNMR(500MHz,CDCl3)δ7.92(d,J=7.8Hz,2H),7.63(t,J=7.4Hz,1H),7.55(t,J=7.6Hz,2H),5.40(d,J=3.8Hz,1H),5.35(t,J=10.0Hz,1H),5.24(t,J=9.2Hz,1H),5.04(t,J=9.8Hz,1H),4.85(dd,J=10.5,3.9Hz,1H),4.79(t,J=8.6Hz,1H),4.53-4.43(m,2H),4.24(d,J=12.1Hz,1H),4.06-3.98(m,2H),3.98-3.91(m,1H),3.80(dt,J=9.0,6.3Hz,1H),3.66(dt,J=9.8,3.6Hz,1H),2.97(t,J=7.3Hz,2H),2.13(s,3H),2.09(s,3H),2.03(s,3H),2.02(s,3H),1.99(s,9H),1.73-1.39(m,4H),1.38-1.12(m,8H).13C NMR(126MHz,CDCl3)δ170.65,170.58,170.37,170.08,169.70,169.53,145.00,133.70,129.37,127.04,100.39,95.61,75.53,72.86,72.32,72.20,70.11,69.45,68.58,68.13,62.97,61.62,36.11,29.33,28.67,28.63,28.48,25.65,21.04,20.98,20.81,20.78,20.73,20.70.高分辨质谱C39H54O20S2[M+Na]+计算值:929.2548;实测值:929.3233。
3g:白色固体,分离产率90%。1H NMR(800MHz,CDCl3)δ7.92(dd,J=8.4,1.2Hz,2H),7.62(t,J=7.4Hz,1H),7.54(t,1H),5.40(d,J=4.0Hz,1H),5.34(dd,J=10.5,9.5Hz,1H),5.23(t,J=9.2Hz,1H),5.04(t,J=9.9Hz,1H),4.84(dd,J=10.5,4.1Hz,1H),4.79(dd,J=9.5,7.9Hz,1H),4.49(d,J=7.9Hz,1H),4.46(dd,J=12.1,2.8Hz,1H),4.25-4.20(m,2H),4.03(dd,J=12.5,2.3Hz,1H),3.98(t,J=9.2Hz,1H),3.95(ddd,J=10.3,3.9,2.3Hz,1H),3.81(dt,J=9.7,6.4Hz,1H),3.65(ddd,J=9.6,4.3,2.8Hz,1H),3.43(dt,J=9.7,6.7Hz,1H),2.97(t,J=7.4Hz,2H),2.12(s,3H),2.08(s,3H),2.03(s,3H),2.01(s,3H),1.99-1.98(m,9H),1.63-1.45(m,4H),1.29-1.18(m,8H).13C NMR(201MHz,CDCl3)δ170.64,170.59,170.37,170.08,169.69,169.53,145.03,133.68,129.36,127.04,100.40,95.62,75.54,72.89,72.33,72.20,70.20,70.12,69.46,68.58,68.15,62.99,61.63,36.15,29.42,29.09,28.91,28.68,28.50,25.77,21.03,20.97,20.80,20.76,20.72,20.69.高分辨质谱C40H56O20S2[M+Na]+计算值:943.2704;实测值:943.2698。
3h:白色固体,分离产率90%。1H NMR(500MHz,CDCl3)δ7.93(d,J=8.0Hz,2H),7.63(t,J=7.4Hz,1H),7.55(t,J=7.7Hz,2H),5.41(d,J=4.0Hz,1H),5.35(t,J=10.0Hz,1H),5.24(t,J=9.1Hz,1H),5.04(t,J=9.8Hz,1H),4.85(dd,J=10.5,3.9Hz,1H),4.80(t,J=8.7Hz,1H),4.50(d,J=8.0Hz,1H),4.46(dd,J=12.2,2.6Hz,1H),4.23(ddd,J=13.2,10.6,4.0Hz,2H),4.05-3.93(m,3H),3.82(dt,J=9.0,6.3Hz,1H),3.66(dt,J=9.3,3.4Hz,1H),3.44(dt,J=9.3,6.8Hz,1H),2.98(t,J=7.2Hz,2H),2.13(s,3H),2.09(s,3H),2.05-1.98(m,15H),1.55(dp,J=m,4H),1.28-1.17(m,10H).13C NMR(126MHz,CDCl3)δ170.68,170.63,170.41,170.11,169.72,169.56,145.02,133.69,129.37,127.05,100.42,95.61,75.56,72.83,72.33,72.17,70.30,70.11,69.45,68.58,68.11,62.99,61.61,36.19,29.46,29.36,29.26,28.95,28.68,28.58,25.86,21.07,21.00,20.83,20.79,20.76,20.73,20.72.高分辨质谱C41H58O20S2[M+Na]+计算值:957.2861;实测值:957.2852。
(iii)2mmol化合物3a-3h溶于10ml无水二氯甲烷中,加入555ul三乙胺(泰坦,货号:01013520),于0℃搅拌10分钟。4mmol对应的硫醇化合物溶解在1ml无水二氯甲烷中,逐滴加入上述反应体系中。于室温再搅拌1小时,旋蒸除掉溶剂。加入20mL乙酸乙酯和20mL水,分出有机相,水相用乙酸乙酯(每次约20ml)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体4a-4h。
其中,结构式分别为4a-4h的中间体对应的硫醇化合物均为市售产品,见下表:
中间体4a-4h结构表征,具体如下:
4a:白色固体,分离产率99%。1H NMR(500MHz,CDCl3)δ5.39(d,J=4.1Hz,1H),5.33(t,J=10.4Hz,1H),5.23(t,J=9.2Hz,1H),5.03(td,J=9.7Hz,1H),4.83(dd,J=10.6,3.9Hz,1H),4.80(dd,J=9.6,8.0Hz,1H),4.55(d,J=7.9Hz,1H),4.46(d,J=12.1Hz,1H),4.22(td,J=12.1,4.3Hz,2H),4.06-4.01(m,2H),3.97(t,J=9.2Hz,1H),3.95-3.92(m,1H),3.79-3.74(m,1H),3.67(q,J=9.8Hz,1H),2.81(t,J=6.6Hz,2H),2.65(t,J=7.4Hz,2H),2.12(s,3H),2.07(s,3H),2.02(s,9H),1.99-1.97(m,6H),1.66-1.61(m,2H),1.34(qd,J=9.0,8.5,4.0Hz,2H),1.30-1.21(m,8H),0.86(t,J=7.1Hz,3H).13C NMR(201MHz,CDCl3)δ170.61,170.53,170.29,170.03,169.78,169.50,100.58,95.62,75.40,72.77,72.28,72.11,70.08,69.43,68.60,68.41,68.12,62.89,61.61,39.25,38.07,31.88,29.27,29.24,29.23,28.59,22.72,20.99,20.94,20.81,20.78,20.70,20.67,20.66,14.19.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2799。
4b:白色固体,分离产率94%。1H NMR(500MHz,CDCl3)δ5.38(d,J=3.6Hz,1H),5.33(t,J=10.0Hz,1H),5.22(t,J=9.0Hz,1H),5.02(t,J=9.8Hz,1H),4.82(dd,J=10.6,3.9Hz,1H),4.78(t,J=8.5Hz,1H),4.49(d,J=7.6Hz,1H),4.45(d,J=11.7Hz,1H),4.21(td,J=11.4,3.8Hz,2H),4.05-3.85(m,4H),3.69-3.55(m,2H),2.66(dq,J=19.3,7.5,6.4Hz,4H),2.12(s,3H),2.07(s,3H),2.03-1.96(m,15H),1.63(p,J=7.6Hz,2H),1.39-1.19(m,10H),0.85(t,J=6.6Hz,3H).13C NMR(126MHz,CDCl3)δ170.61,170.55,170.30,170.02,169.69,169.50,100.44,95.60,75.47,72.80,72.23,72.20,70.07,69.42,68.58,68.10,68.06,62.92,61.59,39.02,34.87,31.78,29.27,28.97,28.80,28.55,22.67,21.00,20.95,20.77,20.69,20.67,14.16.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806实测值:863.2819。
4c:白色固体,分离产率84%。1H NMR(500MHz,CDCl3)δ5.39(d,J=3.7Hz,1H),5.33(td,J=10.0,3.0Hz,1H),5.22(td,J=9.1,2.9Hz,1H),5.03(td,J=9.9,3.3Hz,1H),4.87-4.75(m,2H),4.49(d,J=7.9Hz,1H),4.45(d,J=12.1Hz,0H),4.21(tt,J=13.0,3.5Hz,2H),4.06-3.97(m,2H),3.96-3.90(m,1H),3.86(dd,J=9.8,6.1Hz,1H),3.68-3.62(m,1H),3.49-3.44(m,1H),2.64(td,J=7.2,3.1Hz,4H),2.22-1.94(m,21H),1.65(dtd,J=18.1,11.9,9.3,4.6Hz,6H),1.36(q,J=7.7Hz,2H),1.25(dp,J=16.5,3.8Hz,4H),0.87(t,J=7.0Hz,2H).13C NMR(126MHz,CDCl3)δ170.63,170.58,170.35,170.06,169.72,169.52,100.32,95.59,75.51,72.77,72.24,72.17,70.07,69.57,69.41,68.56,68.08,62.91,61.57,39.15,38.52,31.52,29.25,28.27,28.24,25.63,22.63,21.02,20.97,20.79,20.72,20.68,14.14.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2799。
4d:白色固体,分离产率91%。1H NMR(800MHz,CDCl3)δ5.36(d,J=3.8Hz,1H),5.31(dd,J=10.5,9.5Hz,1H),5.20(t,J=9.2Hz,1H),5.00(t,J=9.9Hz,1H),4.82-4.78(m,1H),4.78-4.73(m,1H),4.47(d,J=7.9Hz,1H),4.42(d,J=12.1Hz,1H),4.22-4.16(m,2H),3.99(d,J=12.5Hz,1H),3.94(t,J=9.2Hz,1H),3.91(dd,J=10.3,2.4Hz,1H),3.83-3.78(m,1H),3.63(dd,J=9.7,2.8Hz,2H),3.46-3.40(m,1H),2.64-2.59(m,4H),2.09(s,3H),2.05(s,3H),1.99(s,3H),1.97(d,J=3.1Hz,6H),1.95(s,6H),1.62(d,J=7.6Hz,4H),1.54(dtt,J=17.4,6.3,2.9Hz,2H),1.40-1.26(m,6H),0.85(t,J=6.9Hz,3H).13C NMR(201MHz,CDCl3)δ170.53,170.48,170.26,169.96,169.61,169.43,100.30,95.54,75.46,72.82,72.23,72.12,70.03,69.86,69.37,68.51,68.07,62.91,61.55,39.11,38.87,30.70,29.06,28.93,28.82,24.80,22.32,20.94,20.88,20.70,20.63,20.60,14.00.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2797。
4e:白色固体,分离产率84%。1H NMR(500MHz,CDCl3)δ5.41(d,J=4.0Hz,1H),5.35(t,J=10.0Hz,1H),5.24(t,J=9.2Hz,1H),5.05(t,J=9.8Hz,1H),4.85(dd,J=10.5,4.1Hz,1H),4.81(dd,J=9.5,7.9Hz,1H),4.50(d,J=7.9Hz,1H),4.47(dd,J=12.2,2.8Hz,1H),4.23(td,J=12.3,4.1Hz,2H),4.05-3.99(m,2H),3.99-3.93(m,1H),3.84(dt,J=9.6,6.3Hz,1H),3.66(ddd,J=9.7,4.4,2.8Hz,1H),3.46(dt,J=9.7,6.7Hz,1H),2.66(h,J=6.4Hz,4H),2.14(s,3H),2.10(s,3H),2.06-1.96(m,15H),1.68-1.62(m,6H),1.61-1.51(m,2H),1.45-1.33(m,4H),0.92(t,J=7.4Hz,3H).13C NMR(126MHz,CDCl3)δ170.72,170.70,170.66,170.43,170.13,169.76,169.58,100.41,95.61,75.57,72.81,72.32,72.18,70.17,70.10,69.46,68.58,68.10,62.98,61.61,39.02,38.92,31.42,29.38,29.20,28.27,25.60,21.79,21.08,21.02,20.85,20.82,20.77,20.74,20.73,13.85.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2800。
4f:白色固体,分离产率99%。1H NMR(800MHz,CDCl3)δ5.39(d,J=4.0Hz,1H),5.34(dd,J=10.5,9.6Hz,1H),5.23(t,J=9.2Hz,1H),5.03(t,J=9.9Hz,1H),4.84(dd,J=10.5,4.0Hz,1H),4.79(dd,J=9.5,7.9Hz,1H),4.49(d,J=7.9Hz,1H),4.45(dd,J=12.1,2.9Hz,1H),4.26-4.19(m,2H),4.02(dd,J=12.5,2.4Hz,1H),3.98(t,J=9.2Hz,1H),3.95(dt,J=10.2,3.2Hz,1H),3.82(dt,J=9.7,6.4Hz,1H),3.65(ddd,J=9.7,4.4,2.9Hz,1H),3.45(dt,J=9.7,6.7Hz,1H),2.67-2.62(m,4H),2.12(s,3H),2.08(s,3H),2.03(s,3H),2.02-1.97(m,12H),1.68(t,J=7.3Hz,2H),1.65(p,J=7.5Hz,2H),1.58-1.50(m,2H),1.38-1.32(m,2H),1.31-1.26(m,4H),0.97(t,J=7.4Hz,3H).13C NMR(201MHz,CDCl3)δ170.64,170.59,170.37,170.07,169.70,169.53,100.40,95.62,75.56,72.89,72.34,72.19,70.21,70.11,69.47,68.58,68.15,63.01,61.63,41.24,39.15,29.42,29.19,29.00,28.52,25.80,22.61,21.03,20.97,20.80,20.76,20.72,20.69,20.68,13.23.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2795。
4g:白色固体,分离产率99%。1H NMR(800MHz,CDCl3)δ5.39(d,J=4.0Hz,1H),5.36-5.31(m,1H),5.24-5.19(m,1H),5.02(dd,J=11.2,8.6Hz,1H),4.83(dd,J=10.5,4.1Hz,1H),4.80-4.76(m,1H),4.48(dd,J=7.9,3.8Hz,1H),4.44(dd,J=12.1,2.8Hz,1H),4.22(td,J=12.3,4.1Hz,2H),4.01(dt,J=12.7,2.3Hz,1H),3.99-3.96(m,1H),3.94(ddd,J=10.3,4.0,2.3Hz,1H),3.81(dt,J=9.7,6.4Hz,1H),3.65(ddd,J=9.6,4.4,2.8Hz,1H),3.44(dt,J=9.7,6.8Hz,1H),2.69-2.63(m,4H),2.11(d,J=2.6Hz,3H),2.07(d,J=4.4Hz,3H),2.02(d,J=2.5Hz,3H),2.00(d,J=2.5Hz,3H),1.99-1.96(m,10H),1.63(p,J=7.4Hz,2H),1.56-1.49(m,2H),1.34(dq,J=12.0,7.2,6.7Hz,2H),1.31-1.24(m,9H).13C NMR(201MHz,CDCl3)δ170.61,170.57,170.34,170.04,169.67,169.50,100.37,95.59,75.54,72.88,72.32,72.15,70.23,70.09,69.44,68.56,68.13,62.99,61.61,39.31,32.88,29.43,29.27,29.23,28.50,25.82,21.01,20.94,20.77,20.73,20.70,20.67,20.66,14.56.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2802。
4h:白色固体,分离产率98%。1H NMR(500MHz,CDCl3)δ5.40(d,J=3.9Hz,1H),5.34(t,J=10.0Hz,1H),5.23(t,J=9.1Hz,1H),5.03(t,J=9.9Hz,1H),4.84(dd,J=10.6,4.1Hz,1H),4.80(t,J=8.6Hz,1H),4.50(d,J=7.8Hz,1H),4.45(dd,J=12.2,2.7Hz,1H),4.23(ddt,J=12.2,8.2,3.6Hz,2H),4.05-3.92(m,3H),3.86-3.78(m,1H),3.66(dd,J=9.2,3.9Hz,1H),3.49-3.41(m,1H),2.68(t,J=7.2Hz,2H),2.39(s,2H),2.13(d,J=3.3Hz,3H),2.09(d,J=3.3Hz,3H),2.03(d,J=3.1Hz,3H),2.02-1.99(m,12H),1.66(p,J=7.6Hz,2H),1.53(p,J=6.8Hz,2H),1.42-1.23(m,10H).13C NMR(126MHz,CDCl3)δ170.65,170.60,170.39,170.08,169.70,169.53,100.41,95.61,75.58,72.88,72.34,72.17,70.31,70.11,69.47,68.58,68.14,63.02,61.63,38.40,29.54,29.48,29.34,29.27,28.58,25.90,23.47,21.05,20.98,20.81,20.77,20.73,20.70.高分辨质谱C36H56O18S2[M+Na]+计算值:863.2806;实测值:863.2801。
(iv)1.8mmol化合物4a-4h溶于10ml甲醇中,加入33ul30%甲醇钠的甲醇溶液(安耐吉,货号:W4200655000),于室温再搅拌1小时。用安伯莱特氢离子交换树脂IR120(百灵威,货号:222221)中和到中性。过滤,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为二氯甲烷∶甲醇=5∶1)纯化分离,冻干后得到二硫键桥连去垢剂(I)-(VIII)。
去垢剂(I)-(VIII)的结构表征,具体如下:
DID-1(I):白色固体,分离产率90%。1HNMR(800MHz,CD3OD)δ5.18(d,J=3.8Hz,1H),4.35(d,J=7.8Hz,1H),4.10(dt,J=10.4,7.0Hz,1H),3.91(dd,J=12.2,2.0Hz,1H),3.89-3.82(m,3H),3.72-3.66(m,2H),3.63(q,J=9.0Hz,2H),3.56(t,J=9.3Hz,1H),3.46(dd,J=9.7,3.8Hz,1H),3.41(ddd,J=9.7,4.7,2.0Hz,1H),3.33(p,J=1.7Hz,2H),3.31-3.24(m,2H),2.95(t,J=7.0Hz,2H),2.75(t,J=7.3Hz,2H),1.71(p,J=7.5Hz,2H),1.43(t,J=7.6Hz,2H),1.38-1.29(m,6H),0.93(t,J=7.0Hz,3H).13C NMR(201MHz,MeOD)δ104.46,102.92,81.23,77.75,76.66,75.07,74.77,74.61,74.16,71.49,69.53,62.75,62.14,39.88,39.09,32.97,30.32,30.30,30.17,29.49,23.70,14.44.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值569.2060。
DID-2(II):白色固体,分离产率90%。1H NMR(500MHz,CD3OD)δ5.14(d,J=3.8Hz,1H),4.26(d,J=7.8Hz,1H),3.96(dt,J=9.8,6.1Hz,1H),3.87(dd,J=12.2,2.1Hz,1H),3.80(ddt,J=8.9,6.8,3.1Hz,2H),3.70-3.55(m,5H),3.52(t,J=9.3Hz,1H),3.43(dd,J=9.7,3.8Hz,1H),3.35(ddd,J=9.6,4.6,2.1Hz,1H),3.28-3.18(m,2H),2.79(t,J=7.2Hz,2H),2.68(t,J=7.2Hz,2H),2.02-1.95(m,2H),1.66(p,J=7.3Hz,2H),1.42-1.32(m,2H),1.30(tt,J=10.9,6.1Hz,7H),0.90(t,J=6.8Hz,2H).13C NMR(126MHz,MeOD)δ104.35,102.91,81.27,77.78,76.56,75.05,74.76,74.64,74.14,71.45,69.04,62.73,62.12,39.57,35.91,32.92,30.40,30.20,30.03,29.45,23.67,14.44.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2061。
DID-3(III):白色固体,分离产率87%。1H NMR(500MHz,CD3OD)δ5.14(d,J=3.8Hz,1H),4.26(d,J=7.8Hz,1H),3.94-3.84(m,2H),3.83-3.76(m,2H),3.69-3.49(m,6H),3.42(dd,J=9.7,3.8Hz,1H),3.35(ddd,J=9.7,4.7,2.1Hz,1H),3.27-3.19(m,2H),2.69(dt,J=17.9,7.1Hz,4H),1.81-1.74(m,2H),1.74-1.68(m,2H),1.68-1.60(m,2H),1.39(p,J=7.2Hz,2H),1.35-1.26(m,4H),0.90(t,J=6.9Hz,3H).13C NMR(126MHz,MeOD)δ104.27,102.92,81.32,77.84,76.57,75.05,74.76,74.66,74.14,71.45,70.27,62.72,62.16,39.69,39.45,32.60,30.18,29.50,29.17,26.81,23.63,14.40.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2062。
DID-4(IV):白色固体,分离产率83%。1H NMR(500MHz,CD3OD)δ5.14(d,J=3.8Hz,1H),4.26(d,J=7.8Hz,1H),3.92-3.77(m,4H),3.69-3.48(m,6H),3.43(dd,J=9.7,3.8Hz,1H),3.35(ddd,J=9.6,4.7,2.1Hz,1H),3.27-3.19(m,2H),2.68(q,J=7.0Hz,4H),1.74-1.59(m,6H),1.53-1.43(m,2H),1.42-1.29(m,4H),0.91(t,J=7.0Hz,3H).13C NMR(126MHz,CD3OD)δ104.30,102.92,81.33,77.84,76.57,75.06,74.77,74.68,74.16,71.46,70.65,62.73,62.16,39.70,39.61,31.72,30.37,30.07,29.94,25.97,23.36,14.34.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2059。
DID-5(V):白色固体,分离产率88%。1H NMR(500MHz,CD3OD)1H NMR(500MHz,CD3OD)δ5.14(d,J=3.8Hz,1H),4.26(d,J=7.8Hz,1H),3.91-3.85(m,2H),3.82-3.77(m,2H),3.72-3.47(m,4H),3.43(dd,J=9.6,3.8Hz,1H),3.35(ddd,J=9.6,4.6,2.0Hz,1H),3.31-3.19(m,2H),2.67(t,J=7.3Hz,4H),1.73-1.47(m,6H),1.45-1.33(m,8H),0.93(t,J=7.4Hz,3H).13C NMR(126MHz,MeOD)δ104.29,102.91,81.31,77.83,76.55,75.05,74.75,74.66,74.14,71.45,70.77,62.72,62.15,39.62,39.42,32.38,30.64,30.17,29.29,26.71,22.57,14.04.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2061。
DID-6(VI):白色固体,分离产率90%。1H NMR(800MHz,CD3OD)δ5.18(d,J=3.8Hz,1H),4.29(d,J=7.8Hz,1H),3.93-3.89(m,2H),3.86-3.82(m,2H),3.72-3.67(m,2H),3.63(td,J=9.2,2.9Hz,2H),3.58-3.54(m,2H),3.46(dd,J=9.7,3.8Hz,1H),3.38(ddd,J=9.7,4.7,2.0Hz,1H),3.29(t,J=9.3Hz,1H),3.25(dd,J=9.4,7.8Hz,1H),2.71(t,J=7.3Hz,2H),2.68(t,J=7.1Hz,2H),1.74-1.69(m,4H),1.65(h,J=7.1Hz,2H),1.47-1.33(m,6H),1.02(t,J=7.4Hz,3H).13C NMR(201MHz,MeOD)δ104.30,102.90,81.33,77.86,76.58,75.07,74.76,74.69,74.16,71.49,70.85,62.75,62.19,41.80,39.73,30.69,30.15,30.12,29.41,26.97,23.44,13.32.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2056。DID-7(VII):白色固体,分离产率92%。1H NMR(800MHz,CD3OD)δ5.18(d,J=3.8Hz,1H),4.29(d,J=7.8Hz,1H),3.94-3.87(m,2H),3.86-3.81(m,2H),3.72-3.66(m,2H),3.63(td,J=9.2,2.7Hz,2H),3.58-3.53(m,2H),3.46(dd,J=9.7,3.8Hz,1H),3.38(ddd,J=9.7,4.7,2.1Hz,1H),3.29(t,J=9.4Hz,1H),3.24(dd,J=9.4,7.8Hz,1H),2.73-2.69(m,4H),1.70(p,J=7.5Hz,2H),1.65(p,J=6.9Hz,2H),1.46-1.39(m,4H),1.39-1.34(m,4H),1.32(t,J=7.3Hz,3H).13C NMR(201MHz,MeOD)δ104.31,102.91,81.33,77.86,76.58,75.07,74.76,74.70,74.16,71.49,70.91,62.75,62.19,39.88,33.39,30.74,30.43,30.26,30.23,29.41,27.02,14.81.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2060。
DID-8(VIII):白色固体,分离产率93%。1H NMR(800MHz,CD3OD)δ5.18(d,J=3.8Hz,1H),4.29(d,J=7.8Hz,1H),3.94-3.88(m,2H),3.87-3.83(m,2H),3.72-3.67(m,2H),3.63(td,J=9.2,2.5Hz,2H),3.55(dd,J=10.3,8.1Hz,2H),3.46(dd,J=9.7,3.8Hz,1H),3.38(ddd,J=9.7,4.7,2.1Hz,1H),3.29(t,J=9.4Hz,1H),3.24(dd,J=9.4,7.8Hz,1H),2.73(t,J=7.3Hz,2H),2.41(s,3H),1.71(p,J=7.4Hz,2H),1.67-1.62(m,2H),1.46-1.30(m,10H).13C NMR(201MHz,MeOD)δ104.31,102.91,81.33,77.86,76.58,75.07,74.76,74.70,74.17,71.49,70.93,62.75,62.19,38.91,30.77,30.57,30.50,30.27,30.19,29.45,27.07,23.32.高分辨质谱C22H42O11S2[M+Na]+计算值:569.2066;实测值:569.2061。
3、去垢剂DID-1-L,DID-3-S和DID-3-L的合成方法如下:
DID-1-L:2mmol化合物3a溶于10ml无水二氯甲烷中,加入555ul三乙胺(泰坦,货号:01013520),0℃搅拌10分钟。4mmol壬硫醇(百灵威,货号:267121)溶解在1ml无水二氯甲烷中,逐滴加入上述反应体系中。于室温再搅拌1小时,旋蒸除掉溶剂。加入20mL乙酸乙酯和20mL水,分出有机相,水相用乙酸乙酯(每次20mL)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体5。中间体5按照2中所述步骤(iv)脱掉乙酰基保护,冻干后得到纯净二硫键去垢剂DID-1-L。
DID-3-S:2mmol化合物3c溶于10ml无水二氯甲烷中,加入555ul三乙胺(泰坦,货号:01013520),0℃搅拌10分钟。4mmol戊硫醇(阿达玛斯,货号:01007922)溶解在1ml无水二氯甲烷中,逐滴加入上述反应体系中。于室温再搅拌1小时,旋蒸除掉溶剂。加入20mL乙酸乙酯和20mL水,分出有机相,水相用乙酸乙酯(每次20mL)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体6。中间体6按照2中所述步骤(iv)脱掉乙酰基保护,冻干后得到纯净二硫键去垢剂DID-3-S。
DID-3-L:2mmol化合物3c溶于10ml无水二氯甲烷中,加入555ul三乙胺(泰坦,货号:01013520),0℃搅拌10分钟。4mmol庚硫醇(TCI,货号:H0029)溶解在1ml无水二氯甲烷中,逐滴加入上述反应体系中。于室温再搅拌1小时,旋蒸除掉溶剂。加入20mL乙酸乙酯和20mL水,分出有机相,水相用乙酸乙酯(每次20mL)萃取三次,并入有机相,用饱和食盐水洗涤,然后用无水硫酸钠干燥,过滤除去硫酸钠,浓缩得到粗产品。粗产品用柱层析(200-300目硅胶,洗脱剂为石油醚∶乙酸乙酯=2∶1)分离得到纯净的中间体7。中间体7按照2中所述步骤(iv)脱掉乙酰基保护,冻干后得到纯净二硫键去垢剂DID-3-L。
对上述化合物进行结构表征,具体如下:
中间体5:白色固体,分离产率89%。δ5.40(d,J=4.0Hz,1H),5.35(dd,J=10.5,9.5Hz,1H),5.25(t,J=9.2Hz,1H),5.04(t,J=9.9Hz,1H),4.88-4.75(m,2H),4.56(d,J=7.9Hz,1H),4.47(dd,J=12.1,2.7Hz,1H),4.23(td,J=12.2,4.1Hz,2H),4.15-3.96(m,3H),3.94(ddd,J=10.3,3.9,2.4Hz,1H),3.78(dt,J=10.5,7.0Hz,1H),3.68(ddd,J=9.7,4.3,2.7Hz,1H),2.82(t,J=6.6Hz,2H),2.66(t,J=7.4Hz,2H),2.14(s,3H),2.09(s,3H),2.04-2.01(m,9H),1.99(s,6H),1.69-1.61(m,2H),1.35(t,J=7.5Hz,2H),1.30-1.21(m,10H),0.87(t,J=6.9Hz,3H).13C NMR(126MHz,CDCl3)δ170.67,170.59,170.35,170.09,169.84,169.55,100.61,95.61,75.41,72.69,72.26,72.10,70.08,69.42,68.59,68.44,68.08,62.87,61.58,39.23,38.03,31.97,29.58,29.37,29.35,29.25,28.62,22.79,21.04,21.00,20.86,20.83,20.75,20.73,20.71,14.25.高分辨质谱C37H58O18S2[M+Na]+计算值:877.2962;实测值:877.2963。
DID-1-L(IX):白色固体,分离产率89%。1H NMR(500MHz,Methanol-d4)δ5.14(d,J=3.8Hz,1H),4.31(d,J=7.7Hz,1H),4.07(dt,J=10.4,7.0Hz,1H),3.90-3.78(m,4H),3.70-3.56(m,4H),3.52(t,J=9.2Hz,1H),3.43(dd,J=9.7,3.8Hz,1H),3.37(ddd,J=9.7,4.6,2.1Hz,1H),3.27-3.19(m,2H),2.91(t,J=7.0Hz,2H),2.71(t,J=7.3Hz,2H),1.67(p,J=7.3Hz,2H),1.38(q,J=6.9,6.5Hz,2H),1.30(q,J=5.2,4.6Hz,10H),0.89(t,J=6.9Hz,3H).13C NMR(126MHz,MeOD)δ104.46,102.94,81.24,77.74,76.64,75.06,74.78,74.60,74.15,71.46,69.53,62.73,62.12,39.84,39.04,33.05,30.63,30.41,30.37,30.17,29.49,23.74,14.46.高分辨质谱C23H44O11S2[M+Na]+计算值:583.2223;实测值:583.2216。
中间体6:白色固体,分离产率80%。1H NMR(500MHz,CDCl3)δ5.38(d,J=3.3Hz,1H),5.32(t,J=9.9Hz,1H),5.21(t,J=9.1Hz,1H),5.01(t,J=9.8Hz,1H),4.85-4.75(m,2H),4.48(d,J=8.0Hz,1H),4.44(dd,J=12.2,2.7Hz,1H),4.20(td,J=13.8,13.1,3.7Hz,2H),4.03-3.90(m,3H),3.87-3.81(m,1H),3.67-3.61(m,1H),3.46(dd,J=10.1,5.9Hz,1H),2.66-2.60(m,4H),2.11(s,3H),2.06(s,3H),2.02-1.95(m,15H),1.72-1.59(m,6H),1.35-1.26(m,4H),0.90-0.84(m,3H).13C NMR(126MHz,CDCl3)δ170.61,170.56,170.32,170.03,169.70,169.49,100.27,95.54,75.46,72.72,72.19,72.12,70.02,69.54,69.36,68.51,68.02,62.86,61.53,39.06,38.47,30.71,28.93,28.20,25.59,22.36,20.99,20.94,20.76,20.69,20.66,20.64,14.05.高分辨质谱C35H54O18S2[M+Na]+计算值:849.2649;实测值:849.2651。
DID-3-S(X):白色固体,分离产率80%。1H NMR(500MHz,Methanol-d4)δ5.17(d,J=3.9Hz,1H),4.28(d,J=7.8Hz,1H),3.96-3.88(m,2H),3.85-3.83(m,1H),3.82-3.80(m,1H),3.72-3.57(m,5H),3.56-3.52(m,1H),3.45(dd,J=9.7,3.8Hz,1H),3.38(ddd,J=9.6,4.6,2.0Hz,1H),3.30-3.19(m,2H),2.77-2.65(m,4H),1.86-1.77(m,2H),1.77-1.72(m,2H),1.71-1.66(m,2H),1.44-1.34(m,4H),0.94(t,J=7.1Hz,3H).13C NMR(126MHz,MeOD)δ132.45,131.07,109.49,106.02,104.76,103.23,102.93,102.84,102.32,99.65,98.43,90.90,90.35,67.86,67.64,59.87,58.09,57.67,54.98,51.51,42.48.高分辨质谱C21H40O11S2[M+Na]+计算值:555.1910;实测值:555.1907。
中间体7:白色固体,分离产率99%。1H NMR(500MHz,CDCl3)δ5.31(d,J=4.1Hz,1H),5.25(td,J=11.4,10.6,4.9Hz,1H),5.14(td,J=9.2,4.0Hz,1H),4.94(td,J=11.9,10.8,5.2Hz,1H),4.73(ddd,J=17.6,9.4,4.4Hz,2H),4.42(d,J=7.7Hz,1H),4.37(dt,J=12.8,3.8Hz,1H),4.18-4.08(m,2H),3.89(ddd,J=28.5,14.4,8.2Hz,3H),3.77(dq,J=9.0,4.9,4.1Hz,1H),3.58(dt,J=10.5,3.6Hz,1H),3.43-3.35(m,1H),2.56(t,J=7.0Hz,4H),2.04(s,3H),1.99(s,3H),1.91(d,J=11.6Hz,15H),1.58(ddt,J=29.3,14.7,7.7Hz,6H),1.26(q,J=6.8Hz,2H),1.22-1.12(m,6H),0.80-0.74(m,3H).13C NMR(126MHz,CDCl3)δ170.38,170.36,170.32,170.11,169.81,169.47,169.28,100.09,95.38,75.28,72.62,72.02,71.95,69.87,69.32,69.18,68.35,67.88,62.72,61.38,38.89,38.27,31.58,29.07,28.78,28.33,28.02,25.43,22.48,20.81,20.75,20.57,20.50,20.46,14.00.高分辨质谱C37H58O18S2[M+Na]+计算值:877.2962;实测值:877.2959。
DID-3-L(XI):白色固体,分离产率83%。1H NMR(500MHz,Methanol-d4)δ5.17(d,J=3.8Hz,1H),4.29(d,J=7.8Hz,1H),3.98-3.87(m,2H),3.87-3.79(m,2H),3.74-3.64(m,2H),3.67-3.59(m,2H),3.62-3.56(m,1H),3.59-3.51(m,1H),3.46(dd,J=9.7,3.8Hz,1H),3.38(ddd,J=9.6,4.8,2.1Hz,1H),3.32-3.21(m,2H),2.73(dt,J=18.5,7.1Hz,4H),1.85-1.65(m,6H),1.42(dddd,J=11.6,9.9,5.8,2.3Hz,2H),1.38-1.26(m,6H),0.98-0.88(m,2H).13C NMR(126MHz,MeOD)δ132.46,131.08,109.50,106.02,104.76,103.23,102.93,102.84,102.33,99.65,98.44,90.91,90.35,67.88,67.64,61.07,58.38,58.19,57.67,57.60,54.98,51.82,42.58.高分辨质谱C23H44O11S2[M+Na]+计算值:583.2223;实测值:583.2212。
实施例2
用实施例1中的各去垢剂形成的胶束性质进行评价
1.CMC测定
各个去垢剂的临界胶束浓度(CMC)采用荧光染料法进行测定。将结构式分别为(I)-(IX)的二硫键桥连去垢剂配成梯度溶液,根据疏水性荧光染料的荧光曲线突变点确定去垢剂的CMC值。例如,用含有40uM荧光染料8-氨基-1-磺酸萘铵盐(百灵威,货号:442848)的双蒸水配制浓度分别为0,42,84,126,168,204,240,360,480,600,800和1000uM的DID-1-L的溶液,避光孵育20分钟。测定477nm/388nm的荧光曲线,曲线突变点便是DID-1-L的CMC值。
2.动态光散射实验
结构式分别为(I)-(IX)的二硫键桥连去垢剂用双蒸水配成0.2%(w/v)的溶液,用动态光散射仪测定各个去垢剂样品胶束的疏水直径(Dh)值,每个样品重复9次。
3.实验结果
从以上结果可以看出,在相同原子个数的基础上,由于二硫键特殊的拓扑结构,实施例1中合成的二硫键去垢剂DID-1-DID-8的CMC值皆表现出上升的趋势。同时,如图2所示,二硫键去垢剂倾向于形成更小体积的胶束。
实施例3
用实施例1中的去垢剂溶解和纯化腺苷受体2A亚型蛋白(A2a蛋白)
1.具体步骤:
在SF9细胞(invitrogen,货号11496-015)中对A2a蛋白进行表达:
细胞在37℃培养至细胞密度达到1.0-1.3x106cells/mL后收集,破碎得到细胞膜。细胞膜用低盐缓冲液(10mM氯化镁(西格玛奥德里奇,货号M4880),20mM氯化钾(西格玛奥德里奇,P9541),pH 7.5的20mMHepes(ABCONE,货号H33755))洗涤(50mL*3次)并离心,再用高盐缓冲液(1M氯化钠(上海生工,货号A610476),10mM氯化镁,20mM氯化钾,20mMHepes,pH7.5)洗涤(50mL*3次)并离心。沉淀物重新悬浮到50ml低盐缓冲液中,加入36mg茶碱(上海生工,货号A500938)和100mg碘乙酰胺(上海生工,货号A600539),于4℃下混匀0.5小时。加入50mL溶膜缓冲液(100mM Hepes,pH 7.5,1.6M氯化钠,200*CMC去垢剂(n-十二烷基-β-D-麦芽糖苷(DDM),Anatrace,货号D310或者结构式分别为(I)-(IX)的二硫键桥连去垢剂),于4℃下混匀3小时,离心得到上清液,加入700mg TALON IMAC树脂(Clontech,货号635670),于4℃下混匀12小时。含有树脂的溶液过滤除去水相,依次用洗涤液1(pH 7.5;800mM氯化钠;10%甘油(西格玛奥德里奇,G5516),20*CMC去垢剂,20mM咪唑(西格玛奥德里奇,货号I5513),10mM氯化镁)、洗涤液2(pH 7.5;500mM氯化钠;10%甘油,10*CMC去垢剂,20mM咪唑)各洗涤3次。用提取液(pH 7.5;300mM氯化钠;10%甘油,5*CMC去垢剂,220mM咪唑)冲洗树脂得到A2a蛋白的缓冲溶液。通过分析凝胶排阻层析(analytical size-exclusionchromatography,aSEC)方法来评价蛋白的产量和样品的均一性。
2.实验结果:
如图3a所示,对于实施例1中合成的二硫键去垢剂可以用于腺苷受体2A亚型蛋白的纯化过程中,其效果与DDM相当或者略差。
实施例4
对实施例3中的去垢剂-A2a复合物进行热稳定性实验。
1.具体步骤:
实施例3得到的A2a蛋白(0.2μM)、荧光染料(Biotium,货号91010)(5μM)混合于缓冲液(pH 7.5;300mM氯化钠;10%甘油,5*CMC去垢剂)中。样品于4℃放置30分钟后,使用Rotor-Gene采集样品的荧光信号(激发波长365nm,发射波长460nm)随温度的变化并绘制曲线,得到该样品的Tm值用于评估配体对蛋白热稳定性的影响。
2.实验结果如下表:
从以上结果可以看出,对于实施例1中合成的大多数二硫键去垢剂对A2a蛋白的热稳定作用略差于DDM,但是像DID-1-L,如图3b所示,其对腺苷受体蛋白的热稳定作用优于DDM。
实施例5
将细菌外膜蛋白OmpX重构于实施例1中的去垢剂中并进行NMR研究
1.具体步骤:
15N标记的OmpX蛋白在大肠杆菌BL-21(诺唯赞,货号C504-03)中标记和表达。细菌在37℃培养至OD600大约为1.0,加入1mM异丙基-β-D-硫代吡喃半乳糖苷(TCI,货号10328)诱导大约5小时后,6000g离心10分钟收集菌体,菌体细胞用TE缓冲液(20mM Tris-HCl,pH=8.0,5mM EDTA(ABCONE,货号E27918)悬浮后,超声破碎菌体细胞。将细菌破碎液离心,菌体沉淀依次用含有2%(v/v)TritonX-100(西格玛,货号:V900502)的TE缓冲液和TE缓冲液洗涤后,离心后得到变性的OmpX蛋白。用含有6M尿素(ABCONE,货号U11681)的TE缓冲液溶解变性的OmpX蛋白,并通过纯化仪(Amersham/GE)进一步纯化。通过3kD浓缩管将蛋白浓缩到12.5mg/mL。取250μL变性的OmpX蛋白溶液缓慢加入到7.5mL折叠缓冲液(50mM Tris-HCl,pH=9,5mM EDTA,1%(w/v)去垢剂)中,室温搅拌16小时。通过浓缩管将溶液置换成NMR缓冲液(20mM PBS,pH=6.8,100mM氯化钠,0.3%叠氮化钠,10%重水(百灵威,货号261750),0.1%(w/v)去垢剂(DDM或者DID-7)),通过Bruker Avance 600MHz核磁仪(BrukerBiospin)测定蛋白样品的2D[15N,1H]-TROSY相关谱。
2.实验结果
如图4所示,溶解在DID-7(图4a)中的细菌外膜蛋白OmpX的2D[15N,1H]-TROSY相关NMR谱图中的核磁信号相对于其在原型去垢剂DDM中的信号(图4b)更为分散清楚,给出了更多的结构信息,从而证实了该类去垢剂在膜蛋白溶液NMR研究中巨大的应用潜力。
Claims (10)
1.一种二硫键桥连的去垢剂分子,其特征在于,其结构式为:
其中,A为未取代的或取代的单糖和取代的二糖,以及各种形式的铵盐中的一种,B为氢或者含有或不含有杂原子的脂肪族取代基;X是含有或不含有杂原子的脂肪族线性亚基。
2.如权利要求1所述的二硫键桥连的去垢剂分子,其特征在于,所述的A为下述结构中的一种:
其中,x为0-3之间的整数,y为1-6之间的整数。
3.如权利要求1所述的二硫键桥连的去垢剂分子,其特征在于,所述的含有或不含有杂原子的脂肪族取代基为下述结构中的一种:
其中m为0-20之间的整数。
4.如权利要求1所述的二硫键桥连的去垢剂分子,其特征在于,所述的X为O(CH2)n,S(CH2)n,NH(CH2)n或NHC(O)(CH2)n,其中n为0-20之间的整数。
5.如权利要求1所述的二硫键桥连的去垢剂分子,其特征在于,所述的A为未取代的二糖,B为直链的脂肪族取代基,X是O(CH2)n,其中n为0-20之间的整数。
6.如权利要求1所述的二硫键桥连的去垢剂分子,其特征在于,其结构式为:
7.权利要求1-6中任一项所述的二硫键桥连的去垢剂分子的制备方法,其特征在于,包括:
步骤1:beta-D-麦芽糖八乙酸酯加入混有分子筛的溶剂中溶解,然后加入第一溴醇化合物;将上述混合物在氮气环境中搅拌,加入三氟化硼乙醚,搅拌过夜;反应结束后,过滤除去分子筛,分出有机相,分离得到第一中间体;
步骤2:将第一中间体溶于溶剂中,加入四丁基溴化铵和硫代苯磺酸钠进行反应,后处理得到第二中间体;
步骤3:将第二中间体溶于溶剂中,加入三乙胺,搅拌,逐滴加入硫醇化合物溶液,搅拌,后处理得到第三中间体;
步骤4:将第三中间体溶于溶剂中,加入甲醇钠的甲醇溶液,搅拌,后处理得到二硫键桥连的去垢剂分子。
8.权利要求1-6中任一项所述的二硫键桥连的去垢剂分子的衍生物,其特征在于,为权利要求1-6中任一项所述的二硫键桥连的去垢剂分子的对映异构体、非对映异构体、几何异构体、互变异构体、旋转异构体、阻转异构体、消旋体、代谢产物、盐类、水合物或高聚物。
9.权利要求1-6所述的二硫键桥连的去垢剂分子或权利要求7所述的二硫键桥连的去垢剂分子的衍生物在膜蛋白分离纯化、离体表达以及结构功能研究的至少一种中的应用。
10.一种去垢剂,其特征在于,其含有权利要求1-6中任一项所述的二硫键桥连的去垢剂分子或权利要求7所述的二硫键桥连的去垢剂分子的衍生物。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108997447A (zh) * | 2018-08-14 | 2018-12-14 | 上海科技大学 | 含有二硫键的化合物及其用途和制备方法 |
CN113880892A (zh) * | 2020-07-03 | 2022-01-04 | 上海科技大学 | 一种小分子去垢剂 |
CN116434828A (zh) * | 2023-04-17 | 2023-07-14 | 深圳新锐基因科技有限公司 | 基于计算结构生物学的蛋白分子动态二硫键的引入方法及装置 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1058229A (zh) * | 1990-01-22 | 1992-01-29 | 普罗格特-甘布尔公司 | 漂白洗涤剂组合物 |
CN104177453A (zh) * | 2014-07-18 | 2014-12-03 | 南京邮电大学 | 一种纳米颗粒表面修饰剂的合成方法 |
-
2017
- 2017-10-19 CN CN201710977131.9A patent/CN107698629B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1058229A (zh) * | 1990-01-22 | 1992-01-29 | 普罗格特-甘布尔公司 | 漂白洗涤剂组合物 |
CN104177453A (zh) * | 2014-07-18 | 2014-12-03 | 南京邮电大学 | 一种纳米颗粒表面修饰剂的合成方法 |
Non-Patent Citations (2)
Title |
---|
KIM LE MAI HOANG,等: "Exploring the Native Chemical Ligation Concept for Highly Stereospecific Glycosylation Reactions", 《J. ORG. CHEM.》 * |
YOSHIKAWA, TAKERU;等: "A highly efficient construction of GM1 epitope tetrasaccharide and its conjugation with KLH", 《GLYCOCONJUGATE JOURNAL》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN108997447A (zh) * | 2018-08-14 | 2018-12-14 | 上海科技大学 | 含有二硫键的化合物及其用途和制备方法 |
CN108997447B (zh) * | 2018-08-14 | 2021-08-24 | 上海科技大学 | 含有二硫键的化合物及其用途和制备方法 |
CN113880892A (zh) * | 2020-07-03 | 2022-01-04 | 上海科技大学 | 一种小分子去垢剂 |
CN113880892B (zh) * | 2020-07-03 | 2023-06-27 | 上海科技大学 | 一种小分子去垢剂 |
CN116434828A (zh) * | 2023-04-17 | 2023-07-14 | 深圳新锐基因科技有限公司 | 基于计算结构生物学的蛋白分子动态二硫键的引入方法及装置 |
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