Synthesis of ferrocenylcarbodiimide as a convenient electrochemically active labeling reagent for nucleic acids Kosuke Mukumoto, b Takahiko Nojima b and Shigeori Takenaka a, * a Department of Materials Science, Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu-shi 804 8550, Japan b Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 812 8581, Japan Received 13 July 2005; revised 12 September 2005; accepted 13 September 2005 Available online 10 October 2005 Abstract—Ferrocenylcarbodiimides carrying different redox potentials, 1 and 2, were designed and synthesized as convenient electrochemically active labeling reagents for nucleic acids, which may be used as dually labeling reagents of nucleic acids like Cy3 and Cy5 dyes. These reagents could react with the imino unit of thymine or guanine base on DNA or of uracil base on RNA under a basic buffer condition to yield a labeled product quantitatively in a short period of time. The current responses of the labeled DNAs in square wave voltammetric (SWV) measurement showed a good linear correlation with the amount of the hybridized ones. DNAs labeled with the two different reagents, 1 and 2, could be detected electrochemically at different potentials after hybridization with a DNA probe-immobilized gold electrode. q 2005 Elsevier Ltd. All rights reserved. 1. Introduction Recently, conventional DNA detecting systems are required in the gene diagnosis and many DNA sensing methods based on electrochemical techniques have been reported, 1–19 as they are expected to realize a convenient gene testing system, which enables direct electronic readout and miniaturization with good cost performance and high sensitivity. Furthermore, they are expected as one of the solutions for the carrier-type gene testing chip and can be applied to various purposes of gene testing such as the point- care test. 20 A variety of electrochemical DNA detecting techniques have been reported by using electrochemically active DNA ligands such as intercalating molecules, 4,7–8,21 or DNA labeling with electrochemically active reagents. 2,5,14–15,19,24–28 Direct electrochemical DNA detection has been achieved under limited conditions using special electrodes and electrolytes. 1,22–23 Ferrocene is often used as an electrochemically active reagent, 2,5,7–9,11,14–15,19,21,24–29 as its reversible redox potential appears where dissolved and atmospheric oxygen does not interfere with the measurement. Oligonucleotides labeled with ferrocene have been used as a DNA probe in electrochemical DNA sensing. Introduction of a ferrocence moiety to oligonucleotides was achieved by a ferrocenyl amide reagent for automated DNA synthesis, 5,19,25–27,30–34 ferrocenyl nucleotide triphosphate as substrate of DNA polymerase, 9,35–38 and the reaction of an activated ester of ferrocene with the amino linked oligonucleotides. 2,11,14,39–41 Direct modification of DNA with ferrocene was also reported by using the Sonogashira reaction of ferrocenyl- propargylamide with halogenated nucleic base of DNA. 42,43 However, all of these methods described here, suffer time- consuming steps and therefore, simpler and more effecient ferrocenylation methods for DNA need to be devised. To achieve a simple labeling method for nucleic acids, ferrocenylcarbodiimide derivatives, 1 and 2, carrying different redox potentials were designed and synthesized. Water-soluble carbodiimide derivatives are known to react with the imino moiety of thymine and guanine bases on DNA or of uracil base on RNA reversibly under basic conditions 44–47 with excellent yield and therefore, ferro- cenylcarbodiimide derivatives should react with DNA and RNA in the same manner, thereby rapidly labeling natural single stranded DNA or RNA fragments with ferrocene as depicted in Scheme 1. Since the redox potential of ferrocene can be altered readily by changing the nature of its substituent, ferrocenylcarbodiimide derivatives having different redox potentials may be prepared by designing a linker connecting ferrocene with carbodiimide parts. Once prepared, such compounds will serve as an important tool to enable competitive analysis of two different samples labeled differentially with the ferrocenyl groups with a different redox potential. 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.09.033 Tetrahedron 61 (2005) 11705–11715 Keywords: Ferrocenylcarbodiimide; Electrochemical detection; DNA; RNA; Dual labeling; Competitive hybridization. * Corresponding author. Tel./fax: C81 93 884 3322; e-mail: shige@che.kyutech.ac.jp