Efficient Synthesis of a Wide‐Range Absorbing Azaphthalocyanine Dark Quencher and Its Application to Dual‐Labeled Oligonucleotide Probes for Quantitative Real‐Time Polymerase Chain Reactions

Abstract

Unsymmetrical dialkylamino-substituted zinc azaphthalocyanine (AzaPc) exhibits unique spectral and photophysical properties for dark quenchers of fluorescence in DNA hybridization probes. The panchromatic light absorption of AzaPc from 300 nm up to at least 700 nm and its lack of fluorescence make it an ideal candidate for a universal dark quencher. To prove this experimentally, oligodeoxyribonucleotide probes were labeled at the 3'-end by this AzaPc and at the 5'-end by a fluorophore used in the polymerase chain reaction (PCR)-that is, fluorescein, CAL Fluor Red 610, and Cy5. AzaPc showed a significantly higher quenching efficiency compared to the commercially available dark quenchers (BHQ-1, BHQ-2, BBQ-650) in a developed model of TaqMan PCR assay. The AzaPc-labeled probe proved to also be useful in a practical PCR assay for the quantification of the SLCO2B1 transporter gene expression. The constructed calibration curves indicated linearity in the range from 10 to 10 of target copies.

Synthesis and J‐Dimer Formation of Tetrapyrazinoporphyrazines with Different Functional Groups for Potential Biomolecular Probe Applications

“…Good solubility of TPyzPz in organic solvents, and specifically polar organic solvents, is essential for efficient binding of the quencher to a polar ODN strand or for the attachment of TPyzPz to a solid phase. In any case, the reaction runs in a heterogeneous system (the ODN to be modified is typically attached to a solid phase), where a high concentration of the reactants increases the chance of successful labeling. Therefore, we used symmetrical TPyzPzs bearing either diethylamino ( 10 , 10H ) or more polar bis(2‐methoxyethyl)amino groups ( 9 , 9H ) as model compounds and compared their solubilities in a series of organic solvents of different polarities (Figure ).…”

“…Advantageously, the hydroxy group of precursor 22 was quantitatively deprotected by attack of excess of alkoxide during cyclotetramerization and provided a free OH group in final TPyzPzs 3 and 7. [18] The different lipophilicities of precursors A and B led to significant differences in the retention factors of particular congeners, thus enabling facile separation of the desired ABBB congener (see photo of a typical TLC plate with a mixture of congeners after statistical condensation, Figure S1, Supporting Information). However, the combination of precursors 22 and 23 gave rise to a mixture of congeners with almost identical retention factors in all tested mobile phases.…”

“…The ESI spectra were recorded in the range 50-5000 m/z using leucineÀ enkefaline as a lock mass reference and sodium iodide for external calibration or in the range 50-1200 m/z using leucineÀ enkefaline as a lock mass reference and sodium formate for external calibration. Following compounds were prepared according to published procedures: TPyzPzs 7H, [8] 7, [8] 9H, [8] 9, [8] 10H, [29] 10, [29] 4-((3-chloro-5,6-dicyanopyrazin-2-yl)(methyl)amino)butanoic acid (13), [12] 5-chloro-6-(diethylamino)pyrazine-2,3-dicarbonitrile (15), [30] 5-chloro-6-(4-(2-hydroxyethyl)piperidin-1-yl)pyrazine-2,3-dicarbonitrile (19), [18] 2-(1-(3chloro-5,6-dicyanopyrazin-2-yl)piperidin-4-yl)ethyl acetate (20), [18] 2-(1-(5,6-dicyano-3-(4-(2-hydroxyethyl)piperidin-1-yl)pyrazin-2-yl)piperidin-4-yl)ethyl acetate (21), [18] 2-(1-(3-(4-(2-azidoethyl)piperidin-1yl)-5,6-dicyanopyrazin-2-yl)piperidin-4-yl)ethyl acetate (22), [18] 5,6bis(bis(2-methoxyethyl)amino)pyrazine-2,3-dicarbonitrile (23), [8] 5,6bis(diethylamino)pyrazine-2,3-dicarbonitrile (24), [31] 4-(methylamino) butanoic acid (29), [16] N,N-di(2-hydroxyethyl)-4-ethynylaniline (30). [17] General synthesis of metal-free TPyzPzs: Precursors A bearing functional groups (i.…”

Emerging Perspectives on Applications of Porphyrinoids for Photodynamic Therapy and Photoinactivation of Microorganisms

“…Very recently, the same group linked oligonucleotides to an unsymmetrical Zn(II)AzaPc at the 3'-end and a fluorophore at the 5'-end for being used as dark quencher too. [42] This bioconjugate showed a large absorption spectrum and excellent quenching efficiencies compared to well-established dark quenchers.…”

The chromatographic behaviour of new double‐labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity

“…The quencher is responsible for quenching the fluorescence emitted by the fluorophore, which could be achieved by static or dynamic processes (Johansson, Fidder, Dick, & Cook, 2002; Lakowicz, 2006). The introduction of dark quenchers, which have no additional fluorescence, simplified the assay and increased its sensitivity owing to the elimination of background noise (Demuth et al, 2018). Unfortunately, to date dark quenchers are mainly based on a few structural motifs and are often suitable only for a restricted number of fluorescent labels owing to the limited spectral overlap of the dyes (Demuth et al, 2018; Kopecky, Novakova, Miletin, Kucera, & Zimcik, 2010).…”