Summary

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Published: March 12, 2015
doi:

Summary

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Abstract

早期检测是治疗成功的关键大多数疾病的,并且是许多类型的癌症的诊断和治疗尤其迫切。所使用的最常见的技术是成像方式如磁共振成像(MRI),正电子发射断层(PET)和计算机辅助断层扫描(CT),并且最佳为了解该疾病的物理结构,但只能每四到执行一次由于使用的成像剂和总成本的六个星期。考虑到这一点,对“点的关怀”技术,如生物传感器,其评估疾病和/或治疗在临床医生的办公室疗效的阶段,做到及时,将彻底改变治疗方案的发展。1作为一种手段来探索基于二茂铁的生物传感器用于生物相关分子2的检测,方法被开发以产生本文所述的二茂铁-生物素的生物共轭物。本报告将重点放在可在金表面被固定生物素二茂铁半胱氨酸系统。

Introduction

生物传感器是采用生物分子识别技术作为用于选择性分析平台和被用于它们的特异性,速度和低成本的小型设备。电化学生物传感器用于生物分子的检测是在这个领域的前沿,由于其简单性,成本效益和高灵敏度。这些传感器1,3的一般解剖是装备有识别分子的电极的特定的感兴趣的生物标志物。该生物标记物的由识别分子结合导致的电位或电流的局部变化可以通过简单的测量来检测。迄今为止,识别部分的范围可以从酶,抗体,4-8,9-12整个细胞,13-16受体,17-2021-23和DNA 24,并 ​​主要集中在大,生物分子25-28研究在这个舞台上的努力主要集中在免疫传感器磨片重的免疫球蛋白是固定有氧化还原活性核(如二茂铁)和用于检测的目标抗体。这些研究被排除在临床应用中,由于精度差,消耗的时间从使用抗原/抗体引起的并发症而产生1,3成长的注意力都集中在小分子检测的生物医学(小于1公斤/摩尔) ,食品和除了国家安全的环境利益。29生物传感器设备的最有名的例子是自测血糖监测仪,其具有耦合到一个口袋大小的安培计丝网印刷酶电极。这些系统通常利用在充电量通过葡萄糖氧化反应产生的总量的测定在一段时间的库仑法。适销对路的设备必须是便携,坚固耐用,手持尽量使用浅显的广大民众。

氧化还原标签,如二茂铁的necessaRY以提供电化学检测生物标志物或小分子的溶液中,因为大多数的生物标志物是无内在电化学活性。30-38二茂是有机金属分子,是金标准电化学,这使得它对于整合到电化学生物传感器的极佳选择。二茂铁基的氧化还原活性种已经获取了相当大的注意,由于它们的尺寸小,稳定性好,方便合成存取,易于化学修饰,相对亲脂性,并且易于氧化还原调谐的。3,30-42小分子基于所述二茂铁芯具有被广泛地用作金属离子和小分子的检测。32-38,43系统靶向较大的品种,如生物分子已经利用大的抗体或免疫球蛋白,以已被嵌入到一个电化学表面二茂铁衍生物的附件。1,3,39 ,44在每一种情况下,该电势和电流的Intensi的铁 / 铁氧化还原对TY被改变时分子偶联,从而产生一个新的分光手柄,指示所述分析物分子的存在。这种变化源于该环戊二烯基环的pI系统和铁d轨道之间产生的大量重叠。如果PI-系统被修改, 衍生或反应,则该轨道相互作用将反过来,变化。这将影响到铁芯,并且可以观察到在的铁III /铁夫妇的电位的移位。40,45,46这些性质使得用作定量剂在电化学免疫测定或生物传感器这种系统的吸引力。

为了产生特异于生物传感器的能力含二茂铁的系统是最佳​​的修改一个Cp环与生物受体特异于靶分子,并利用其它的Cp环作为分子系绳到电化学读数或ELEC处接上( 图1)。这些不对称二茂铁衍生物的合成是由副反应并在分子间交联形成的二聚和聚合物质的形成了挑战。47然而,偶联化学制备的酰胺键是最直接的途径,以提供二茂铁的涉及生物组分,例如简单的衍生物肽和它们的代谢物。因此,首先在20世纪50年代开发的Merrifield用于肽合成的固相技术可以应用到含二茂铁的有机金属化合物。通过使用正交取代的1'-Fmoc-氨基二茂铁-1-羧酸分子,二茂铁系统,它可以包含一个受体部分(生物素),电化学读数(二茂铁),并固定化 – 连接体组分(半胱氨酸)的具有已建成和在此详述。此生物缀合物的合成进行了讨论,以及证据固定在金表面上。这项工作represenTS生物素,二茂铁和氨基酸用于固定在金表面的组成的系统的第一表示。

Protocol

1.合成生物素的Fc半胱氨酸(1) 固相方法,以产生树脂结合的1。 放置生物素加载树脂(250毫克,0.145毫摩尔)转化为烧结注射器和通过制定二甲基甲酰胺(5毫升)中,并摇动在实验室摇动器在注射器20分钟使树脂溶胀。排出溶液和二甲基甲酰胺重复肿胀一次。 通过加入4-6毫升20%哌啶的二甲基甲酰胺在注射器随后振荡10-15分钟,除去Fmoc保护基。与另一4-6毫升哌啶…

Representative Results

1,树脂结合形式示于图2。二茂铁组分的共价连接产生了一种橙色色调的树脂珠粒是持久连续洗涤和表示含有复杂的固定化铁如由相对于铁的吸收树脂珠粒的PEG部分。的1对无树脂形式是彩色的,以使树脂珠粒相同。以下除去从树脂珠的化合物,纯度和产率(68%),从所述方法得到的是远远优于典型的解决方案的方法。产品的元素分析表明,1被?…

Discussion

不对称二茂铁衍生物的合成是具有挑战性的溶液。例如,曾试图以产生1的溶液导致期望的产物(低于20%)的产量低。同样地,利用1'-氨基二茂铁羧酸没有的Fmoc)和树脂结合的生物素的反应导致与报道Baristic 经聚合的产物一致不溶产物。和最小的产品。47这是通过二茂铁进一步复杂化和其衍生物是光敏感的,该氨基同源容易在溶液中二聚化。这些问题使?…

Declarações

The authors have nothing to disclose.

Acknowledgements

KG是由RA韦尔奇基金会授予P-1760支持,数学和科学教育(以KG)的TCU安卓研究所,TCU研究和创新活动格兰特(以KG)和TCU电监会格兰特(以JHS)。

Materials

Biotin Novatag Resin NovaBiochem 8550510001
TORVIQ 10ML LUER LOCK FRITTED SYRINGE Fisher NC9299151
piperdine Acros P/3520/PB05
ninhydrin test Sigma-Aldrich 60017-1ea
1’-Fmoc-amino-ferrocene-1-carboxylic acid Omm Scientific Special Order
1-​Hydroxybenzotriazole hydrate Sigma-Aldrich 157260-25G
N,N′-Diisopropylcarbodiimide Sigma-Aldrich D125407-5G
N,N-​Diisopropylethylamine Sigma-Aldrich 496219-100ML
Fmoc-Cys(Trt)-OH Novabiochem 8520080025
trifluoroacetic acid Sigma-Aldrich T5408
1,2-ethanedithiol Sigma-Aldrich 2930
triisopropyl silane Sigma-Aldrich 233781
Eppendorf tubes (20 mL) any source
methanol any source dry with molecular sieves prior to use & store in 100 mL media bottle for easy usage.
dichloromethane any source dry with molecular sieves prior to use & store in 100 mL media bottle for easy usage.
dimethylformamide any source dry with molecular sieves prior to use & store in 100 mL media bottle for easy usage.
centrifuge any source

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Scarborough, J. H., Gonzalez, P., Rodich, S., Green, K. N. Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology. J. Vis. Exp. (97), e52399, doi:10.3791/52399 (2015).

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