固相有机合成与裂解反应动力学研究
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摘要
组合化学是根据组合原理在短时间内将不同构建模块以共价键系统地、反复地进行连接,从而产生大批的分子多样性群体,形成化合物库(Compound Library);然后,运用组合原理,以巧妙的手段对化合物库进行筛选、优化,得到可能的有目标性能的化合物结构的科学,是一门将化学合成、组合理论、计算机辅助设计及机器人结合为一体的技术。
就合成技术而言,组合合成的方法包括固相合成和液相合成两种。就合成方法来说,组合合成分为混合均分法、平行合成法以及混合试剂合成法等。
固相载体上合成多种小分子有机化合物库在组合化学研究领域已成为非常重要的一项技术。其关键组成部分,固相载体与化合物的连接分子linker在固相有机合成(SPOS)上起着非常重要的作用。在从酸敏感linker上裂解化合物的时候,一般常用的裂解试剂是三氟乙酸(TFA)溶液。然而三氟乙酸在使用蒸发方法将其从最终产物中除去时是难以实现完全消除的。残留的TFA存留在产物中,经过贮存过程以及溶剂的挥发,使之浓度升高,这种高浓度的酸可能会使产品分解变质。还有研究发现,10-100nM浓度的TFA作用于胎鼠的造骨细胞,在24h后能抑制细胞增殖以及减少胸腺嘧啶脱氧核苷的生成。在对关节软骨细胞和幼鼠头盖骨成骨细胞的培养过程中也发现了类似的结果,表明TFA并不只对一种细胞有毒性。所有这些发现表明在高通量筛选中,残留的微量TFA能引起严重的假阳性或假阴性的结果,因此必须寻找TFA的替代物来解决这一问题。相比之下,作为一种生物兼容性比较好的试剂,HCI比较容易从产物中除去,利用简单挥发的方法就可实现。即使在产物中有残留,也不会降解产物或对以后的生物活性筛选产生影响。因此,如果盐酸溶液可以进行这种酸敏感linker的裂解反应以取代TFA,将是一个非常好的裂解试剂,对树脂的再生也有重要的意义。本课题通过监测动力学过程,首次详细研究HC1作为裂解试剂在固相有机合成裂解反应中的应用。
官能团的转化在有机合成反应中占有非常重要的地位,它不仅关系到合成步骤的选择,还直接影响产物的纯度以及产率。Mitsunobu反应是有机合成中比较常用的用来进行官能团转换的反应,因为它的反应条件比较温和,使反应较易进行。它是在三苯基磷(Ph_3P)和偶氮二碳酸二乙酯(DEAD)或偶氮二碳酸二异丙酯(DIAD)作用下,伯醇、仲醇与酚发生亲核取代反应,称为Mitsunobu偶联反应,是生成混合醚的一种重要方法。Mitsunobu反应已经被证明是一个有效形成C-O,C-N,C-C,C-X键的方法。其优点是:反应条件温和;立体选择性好;适用于带各种官能团的底物。反应条件温和以及很好的立体选择性使它成为有机合成上非常重要的合成方法,在组合合成上有很高的利用度。尽管如此,Mitsunobu反应也有一些局限,比如通过此反应制得醚类化合物的反应产率不是很高(文献报道在60%-80%居多),而且纯化的方法对化合物库的合成不太现实。
傅立叶变换红外光谱(FTIR)仪是现代化学试验最常规的分析仪器之一,广泛用于有机合成的定性及定量分析,近年来FTIR分析及其新技术在SPOS中的应用非常广泛。FTIR分析可以很方便地用于固相有机反应研究的各个阶段,包括表征固载化合物、实时跟踪检测反应进程、固相反应动力学研究等。与TLC在液相分析中的作用一样,FTIR已经成为固相有机化学中最便利、最实用的分析方法。
Combinatorial chemistry, as an enabling discovery technology, was widely implemented in drug discovery and other research disciplines in the 90s of last century. Synthesis of diverse organic compound libraries on solid supports has been a key methodology in combinatorial chemistry as applied for drug discovery. One crucial component of solid-phase organic synthesis (SPOS) is the linker strategy. Various linkers, such as acid-, base-, or photo-cleavable linkers, have been developed to accommodate synthesis and the cleavage reactions. Among various linkers and cleavage strategies, the application of acid-labile linkers that are cleaved by trifluoroacetic acid (TFA) is probably the most popular approach. However, TFA is difficult to remove completely in the final products by available evaporation methods. The residual amount TFA in the product can become highly concentrated when the compound is stored as stock solution or dried compound. In the presence of TFA. compounds with acid sensitive scaffolds are degraded with time. Furthermore, TFA at a concentration of 10 to 100 nM reduced cell numbers and some research showed that the toxic effect of TFA is not specific to one cell type or to one species of origin. When the activities of the TFA and HCl were compared in osteoblasts, cell proliferation was consistently less with TFA, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding indicated that TFA may cause serious false positive or false negative problems in cell-based high-throughput screening and HC1, on the other hand, is a biologically compatible reagent.
In this work, we chose 18 solid phase compounds to explore the feasibility of using HC1 as an alternative in acid cleavage reactions. HC1 is very easy to remove completely from samples and it does not cause toxic effect in cell assays. In this investigation, we studied the cleavage reaction kinetics of an array of diverse compounds using single bead FTIR microspectroscopy. These compounds were linked to 2% PS-DVB resins through three commonly used linkers at different HC1 concentrations. Most compounds studied in this work can be easily cleaved using low concentration of HC1 (0.9-2.3%) and less time (60-90 min). Therefore, our kinetics studies established HC1 as a biocompatible, removable and effective substitute for TFA when final compounds are used for biological screening and drug discovery.
Transform of functional groups plays an important role in organic chemistry which we often use in synthetic process. The Mitsunobu reaction is extensively used in organic synthesis under mild conditions for the functionalization of alcohols and related compounds. It is easy to purify the products than the solution-phase reactions because the phosphide can be removed by filtration from the polymer-bound products. Solid phase Mitsunobu ether formation can be used in the design and synthesis of combinatorial libraries in many different ways. However, ether formation via Mitsunobu reaction was complicated by low yields, the need for chromatographic purification made this route impractical for library generation. Besides the conditions they used are varied, it is not surprising that almost all of the solid-phase experimental protocols described to date advocate different optimal reaction conditions. Here we studied the optimized process between Wang resin and a phenol through its yields and kinetic investigation, also we applied it to a set of phenols. Finally we found that most phenols could react with Wang resin except one whose LogP is very big. Moreover we found that spatial hindrance on phenols has no effect.
FTIR microspectrscopy technology has been used in monitoring solid phase organic synthesis (SPOS), it is very popular because it is very convenient, fast and simple. It can be used to determination of the presence and the loadings of functional groups and confirm the occurrence and completion of reactions on resins. It can support in shortening the solid-phase synthesis optimization time consuming in combinatorial chemistry. This technique allows the differentiation of functional groups of resin-bound compounds with minor structural differences. In this study, we have applied this technique to the determination of product identity and reaction time course on solid supports by analyzing a single resin bead.
就合成技术而言,组合合成的方法包括固相合成和液相合成两种。就合成方法来说,组合合成分为混合均分法、平行合成法以及混合试剂合成法等。
固相载体上合成多种小分子有机化合物库在组合化学研究领域已成为非常重要的一项技术。其关键组成部分,固相载体与化合物的连接分子linker在固相有机合成(SPOS)上起着非常重要的作用。在从酸敏感linker上裂解化合物的时候,一般常用的裂解试剂是三氟乙酸(TFA)溶液。然而三氟乙酸在使用蒸发方法将其从最终产物中除去时是难以实现完全消除的。残留的TFA存留在产物中,经过贮存过程以及溶剂的挥发,使之浓度升高,这种高浓度的酸可能会使产品分解变质。还有研究发现,10-100nM浓度的TFA作用于胎鼠的造骨细胞,在24h后能抑制细胞增殖以及减少胸腺嘧啶脱氧核苷的生成。在对关节软骨细胞和幼鼠头盖骨成骨细胞的培养过程中也发现了类似的结果,表明TFA并不只对一种细胞有毒性。所有这些发现表明在高通量筛选中,残留的微量TFA能引起严重的假阳性或假阴性的结果,因此必须寻找TFA的替代物来解决这一问题。相比之下,作为一种生物兼容性比较好的试剂,HCI比较容易从产物中除去,利用简单挥发的方法就可实现。即使在产物中有残留,也不会降解产物或对以后的生物活性筛选产生影响。因此,如果盐酸溶液可以进行这种酸敏感linker的裂解反应以取代TFA,将是一个非常好的裂解试剂,对树脂的再生也有重要的意义。本课题通过监测动力学过程,首次详细研究HC1作为裂解试剂在固相有机合成裂解反应中的应用。
官能团的转化在有机合成反应中占有非常重要的地位,它不仅关系到合成步骤的选择,还直接影响产物的纯度以及产率。Mitsunobu反应是有机合成中比较常用的用来进行官能团转换的反应,因为它的反应条件比较温和,使反应较易进行。它是在三苯基磷(Ph_3P)和偶氮二碳酸二乙酯(DEAD)或偶氮二碳酸二异丙酯(DIAD)作用下,伯醇、仲醇与酚发生亲核取代反应,称为Mitsunobu偶联反应,是生成混合醚的一种重要方法。Mitsunobu反应已经被证明是一个有效形成C-O,C-N,C-C,C-X键的方法。其优点是:反应条件温和;立体选择性好;适用于带各种官能团的底物。反应条件温和以及很好的立体选择性使它成为有机合成上非常重要的合成方法,在组合合成上有很高的利用度。尽管如此,Mitsunobu反应也有一些局限,比如通过此反应制得醚类化合物的反应产率不是很高(文献报道在60%-80%居多),而且纯化的方法对化合物库的合成不太现实。
傅立叶变换红外光谱(FTIR)仪是现代化学试验最常规的分析仪器之一,广泛用于有机合成的定性及定量分析,近年来FTIR分析及其新技术在SPOS中的应用非常广泛。FTIR分析可以很方便地用于固相有机反应研究的各个阶段,包括表征固载化合物、实时跟踪检测反应进程、固相反应动力学研究等。与TLC在液相分析中的作用一样,FTIR已经成为固相有机化学中最便利、最实用的分析方法。
Combinatorial chemistry, as an enabling discovery technology, was widely implemented in drug discovery and other research disciplines in the 90s of last century. Synthesis of diverse organic compound libraries on solid supports has been a key methodology in combinatorial chemistry as applied for drug discovery. One crucial component of solid-phase organic synthesis (SPOS) is the linker strategy. Various linkers, such as acid-, base-, or photo-cleavable linkers, have been developed to accommodate synthesis and the cleavage reactions. Among various linkers and cleavage strategies, the application of acid-labile linkers that are cleaved by trifluoroacetic acid (TFA) is probably the most popular approach. However, TFA is difficult to remove completely in the final products by available evaporation methods. The residual amount TFA in the product can become highly concentrated when the compound is stored as stock solution or dried compound. In the presence of TFA. compounds with acid sensitive scaffolds are degraded with time. Furthermore, TFA at a concentration of 10 to 100 nM reduced cell numbers and some research showed that the toxic effect of TFA is not specific to one cell type or to one species of origin. When the activities of the TFA and HCl were compared in osteoblasts, cell proliferation was consistently less with TFA, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding indicated that TFA may cause serious false positive or false negative problems in cell-based high-throughput screening and HC1, on the other hand, is a biologically compatible reagent.
In this work, we chose 18 solid phase compounds to explore the feasibility of using HC1 as an alternative in acid cleavage reactions. HC1 is very easy to remove completely from samples and it does not cause toxic effect in cell assays. In this investigation, we studied the cleavage reaction kinetics of an array of diverse compounds using single bead FTIR microspectroscopy. These compounds were linked to 2% PS-DVB resins through three commonly used linkers at different HC1 concentrations. Most compounds studied in this work can be easily cleaved using low concentration of HC1 (0.9-2.3%) and less time (60-90 min). Therefore, our kinetics studies established HC1 as a biocompatible, removable and effective substitute for TFA when final compounds are used for biological screening and drug discovery.
Transform of functional groups plays an important role in organic chemistry which we often use in synthetic process. The Mitsunobu reaction is extensively used in organic synthesis under mild conditions for the functionalization of alcohols and related compounds. It is easy to purify the products than the solution-phase reactions because the phosphide can be removed by filtration from the polymer-bound products. Solid phase Mitsunobu ether formation can be used in the design and synthesis of combinatorial libraries in many different ways. However, ether formation via Mitsunobu reaction was complicated by low yields, the need for chromatographic purification made this route impractical for library generation. Besides the conditions they used are varied, it is not surprising that almost all of the solid-phase experimental protocols described to date advocate different optimal reaction conditions. Here we studied the optimized process between Wang resin and a phenol through its yields and kinetic investigation, also we applied it to a set of phenols. Finally we found that most phenols could react with Wang resin except one whose LogP is very big. Moreover we found that spatial hindrance on phenols has no effect.
FTIR microspectrscopy technology has been used in monitoring solid phase organic synthesis (SPOS), it is very popular because it is very convenient, fast and simple. It can be used to determination of the presence and the loadings of functional groups and confirm the occurrence and completion of reactions on resins. It can support in shortening the solid-phase synthesis optimization time consuming in combinatorial chemistry. This technique allows the differentiation of functional groups of resin-bound compounds with minor structural differences. In this study, we have applied this technique to the determination of product identity and reaction time course on solid supports by analyzing a single resin bead.
引文
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[3]王德心 寻找新药的捷径--组合化学 中国新药杂志 1997,6:323
[4]董安钢 唐颐 浅谈组合化学 大学化学 2000,15:8
[5]王志宏 组合化学概述 天津药学 2004,16:64
[6]周南 组合化学概述 上海化工 1999,20:4
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[2]Geysen H.M.,Moluen R.H.and Batteling S.J.,Use of peptide synthesis to probe viral antigens fo repitopes to aresolution of a single amino acid Proc.Natl.Acad.Sci.USA,1984,81(13):3998
[3]王德心 寻找新药的捷径--组合化学 中国新药杂志 1997,6:323
[4]董安钢 唐颐 浅谈组合化学 大学化学 2000,15:8
[5]王志宏 组合化学概述 天津药学 2004,16:64
[6]周南 组合化学概述 上海化工 1999,20:4
[7]Furka A.,Seheatyen F.,Augedom M.,et al.General method for rapid synthesis of multicomponent peptide mixtures.Int.J.peptide Protein Res.,1991,37:487
[8]Zuckermann R.N.,Kerr J.M.,Kent S.B.H.and Moos W.H.Efficient method for the preparation of peptoids oligo(N-substituted glycines)by submonomer solid-phase synthesis J.Am.Chem.Soc.1992,114:10646
[9]Gray N.S.,Kwon S.and Schultz P.G.Combinatorial synthesis of 2,9-substituted purines Tetrahedron Lett.1997,38:1161
[10]Chen C.X.,Randall L.A.,R.Miller B.,Jones A.D.and Kurth M.J."Analogous" organic synthesis of small-compound libraries:validation of combinatorial chemistry in small-molecule synthesis J.Am.Chem.Soc.;1994,116:2661
[11]Hiroshige M.,Hauske J.R.and Zhou P.Formation of C--C bond in solid phase synthesis using the heck reaction Tetrahedron Lett.1995, 36: 4567
[12] Dolle, R. E., Comprehensive survey of combinatorial library synthesis J. Comb. Chem. 2005, 8: 597
[13] 李伟章 恽榴红 固相有机合成研究进展 有机化学 1998, 18:403
[14]Chechik V., Crooks R. M. Dendrimer-encapsulated Pd nanoparticles as fluorous phase-soluble catalysts J. Am. Chem. Soc.,2000, 122: 1243
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