Synthesis of mono-, di-, tri- and tetracarboxy azaphthalocyanines as potential dark quenchers Kamil Kopecky a , Dalibor  Satinský b , Veronika Novakova c , Miroslav Miletin a , Antonín Svoboda b , Petr Zimcik a, * a Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic b Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic c Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic article info Article history: Received 10 December 2010 Received in revised form 7 March 2011 Accepted 11 March 2011 Available online 21 March 2011 Keywords: Dark quencher Fluorescence HPLC Phthalocyanine Singlet oxygen Tetrapyrazinoporphyrazine abstract Mono-, di-, tri- and tetracarboxy-substituted metal-free azaphthalocyanines (AzaPc) were synthesized from 5,6-bis(diethylamino)pyrazine-2,3-dicarbonitrile and 6-(5,6-dicyano-3-(diethylamino)pyrazin-2- ylamino)hexanoic acid using a statistical condensation approach. AzaPc bearing eight diethylamino peripheral substituents was also isolated from the mixture. Analysis of the distribution of congeners in the statistical mixture using optimized HPLC method (Phenomenex Synergy RP Fusion column, aceto- nitrile/tetrahydrofuran/water (pH 5.5) 50:20:30) was performed. The analysis showed optimal ratios of starting materials to be 3:1 for AAAB, 1:3 for ABBB and 1:1 for AABB/ABAB types of the congeners. The distribution of the congeners corresponded well with calculated values indicating similar reactivity of both starting materials and no sterical constraint between adjacent isoindole units in the AzaPc ring. All investigated AzaPc showed no fluorescence, extremely low singlet oxygen quantum yields (F Δ < 0.005) in monomeric form and strong absorption in a wide range from 300 nm to almost 700 nm. Such properties are highly promising for future investigation of these compounds as dark quenchers of fluorescence in DNA hybridization probes. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Azaphthalocyanines (AzaPc) are aza-analogues of the well known and widely-investigated synthetic dyes, the phthalocyanines (Pc). The large, planar macrocyclic system of these dyes is a reason for interesting properties utilized in several applications connected either with light absorption, electron transfer or oxidation and reduction. Consequently AzaPc were recently investigated as promising non-linear optical limiting dyes [1,2] or as potential photosensitizers in photodynamic therapy [3e5]. Their fluorescence [6,7], acid-base [8,9], electron-deficient [10e13], aggregating and non-aggregating [14,15] and microporous [16] properties have also been reported. Our recent efforts have been aimed at the investi- gation of AzaPc dyes in a completely new application. When suitably substituted, they are able to quench the fluorescence of different fluorophores in DNA hybridization probes and they were shown to be highly efficient as modern type of non-emitting quenchers, so called “dark quenchers” [17]. The use of DNA-hybridization probes that generate fluorescence only when they bind to their targets enables real-time monitoring of the polymerase chain reaction and is widely used in genetic analysis [18]. The method is based on presence of two dyes e a fluorophore and a quencher, the latter is able to quench fluorescence of the former when they are placed close to each other in space. A broad absorption spectrum, no intrinsic fluorescence and very low singlet oxygen production in the mono- meric form are highly desirable for quenchers in this application. These conditions are met for (dialkylamino)- substituted AzaPc. Besides typical B- and Q-bands in the regions of 360 nm and 650 nm, respectively, they also absorb strongly around 500 nm due to the participation of the amine lone pairs in conjugation with the AzaPc core. Moreover, excited states of (dialkylamino)-AzaPc are efficiently deactivated by ultrafast intramolecular charge transfer (ICT) from the lone pairs of the peripheral dialkylamino groups (donors) to the macrocyclic system (acceptor) [19]. As a consequence of ICT, no fluorescence occurs and the singlet oxygen quantum yield is extremely low. The first AzaPc that were investigated as dark quenchers in DNA hybridization probes contained only one functional group, through * Corresponding author. Tel.: þ420 495067257; fax: þ420 495067167. E-mail address: petr.zimcik@faf.cuni.cz (P. Zimcik). Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig 0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dyepig.2011.03.016 Dyes and Pigments 91 (2011) 112e119