Development and application of a novel acridinium ester for use as a chemiluminescent emitter in nucleic acid hybridisation assays using chemiluminescence quenching

Abstract

Chemiluminescent acridinium esters (AEs) permit the development of high sensitivity ligand binding assays due to a combination of high intensity light emission and very low backgrounds. Here these advantages are exploited for use in homogeneous nucleic acid hybridisation assays using quenched chemiluminescence. AE chemiluminescence is conventionally initiated at highly alkaline pH. Novel "active" AEs were designed that permit initiation under conditions compatible with maintenance of nucleic acid hybrids (i.e. pH less than 9). Methyl red was found to be a dark quencher species capable of functioning at this pH. Practical application of the chemiluminescence quenching assay system has been demonstrated using two model nucleic acid hybridisation assays based on intra- and intermolecular emitter/quencher pairs.

A green synthesis of chemiluminescent N-sulfopropyl acridinium esters in ionic liquids without using the carcinogen 1,3-propane sultone

“…Synthetic access to such compounds is very difficult owing to the poor reactivity of the acridine nitrogen. For example, Brown and coworkers [36] recently described the synthesis of acridinium esters containing long chain N-alkyl groups by N-alkylation of the acridine ester precursors using iodide or trifluoromethanesulfonate alkylating reagents. Typical reported yields were B15% (3Á14%) using these powerful alkylating reagents and, moreover, these N-alkylation reactions were conducted in toxic solvents such as nitrobenzene or 1,2-dichloroethane.…”

9-(4-Fluorophenoxycarbonyl)-10-methylacridinium trifluoromethanesulfonate

“…The dihedral angle is that between the planes of the rings I and J. CgI_Perp is the perpendicular distance of CgI from ring J. CgI_Offset is the distance between CgI and perpendicular projection of CgJ on ring I. Data collection: CrysAlis CCD (Oxford Diffraction, 2008); cell refinement: CrysAlis RED (Oxford Diffraction, 2008); data reduction: CrysAlis RED; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 (Farrugia, 1997); software used to prepare material for publication: SHELXL97 and PLATON (Spek, 2009 9-(4-Fluorophenoxycarbonyl)-10-methylacridinium trifluoromethanesulfonate D. Trzybinski, K. Krzyminski and J. Blazejowski Comment 9-(Phenoxycarbonyl)-10-methylacridinium salts have long been known as chemiluminescent indicators or the chemiluminogenic fragments of chemiluminescent labels widely used in assays of biologically and environmentally important entities such as antigens, antibodies, enzymes or DNA fragments (Zomer & Jacquemijns, 2001;Roda et al, 2003;King et al, 2007;Brown et al, 2009). The cations of these salts are oxidized with hydrogen peroxide in alkaline media, which produces light.…”

“…For general background to the chemiluminogenic properties of 9-phenoxycarbonyl-10-methylacridinium trifluoromethanesulfonates, see: Brown et al (2009); King et al (2007); Rak et al (1999); Roda et al (2003); Zomer & Jacquemijns (2001). For related structures, see: Sikorski et al (2005); Trzybiń ski et al (2010).…”

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

“…A seminal study by Yang et al showed that using a three quencher assembly instead of a single quencher significantly increased the S:N ratio of their MB design from 14 to 320 [13]. Besides the improvement of quenching efficiency, another strategy that has proven successful at reducing F close is the use of a chemiluminescent detection approach with a greatly reduced background signal [14–15]. Chemiluminescent MBs have been able to achieve lower detection limits in the femto to attomolar range, which is ~4 orders of magnitude more sensitive than typical fluorescent MB probes [14].…”