Fig 1.
The transformation of hexachlorofluorescein dye (I) with aqueous ammonia and the next photochemical arylacridine transformation.
R = 5'-oligodeoxyribonucleotide-spacer
Fig 2.
The curves of fluorescence accumulation (CFAs) obtained for H38B2 and A38B2 probes (see S2 Table).
The curves obtained with: (A) pure probe, (B) artificial mixture (3:1, mol/mol) of H38B2 with its acridine analog A38B2, (C) pure A38B2. The amounts of amplicone were equal in every analysis, judjing by electrophoresis data as so as mentioned in [21].
Table 1.
Optical characteristics of HEX- and ACR-fluorophores.
Fig 3.
Spectra of mixtures of HEX-T10 (HT10) with different molar percentages of its arylhexachloroacridine analog (AT10).
(A) VIS- and (B) fluorescent spectra in 0.1 M ammonium acetate, pH 6.7, 20% MeCN, 45°C. Baselines of spectra are moved apart to separate the curves.
Fig 4.
HPLC profiles of (A) 5'-FAM-27mer-Dabs-3' (F27D) and (B) 5'-DMTr-d(ACGT)3-Dabs-3' ODNs (12D). (C) The structure of Dabs-3'-CPG.
Fig 5.
HPLC preparative profiles of several HEX-oligonucleotides.
(A) After standard ammonolysis. (B) Obtained by new deblocking method. Grey filling up here and further marks ACR-containing zones and the black one corresponds to the pure HEX-ODNs fractions.
Fig 6.
VIS-spectra of artificial mixtures of hexachloroflurescein-ODNs with different molar percentage of their hexachloroarylacridine analogs.
(A) HEX-BHQ1. (B) HEX-BHQ2. (C) HEX-Dabs. 0.1 M ammonium acetate, pH 6.7, 10–20% MeCN, 45°C. Baselines of spectra are moved apart to separate the curves.
Fig 7.
D500 / D540 ratios dependence from molar content of ACR-derivatives in HEX-oligonucleotides solutes.
Conditions are as for Fig 6. The types of quencher are shown and the inscription "No" marks quencher-free ODN.
Fig 8.
HPLC profile of HT10 ODN after K2CO3/MeOH deblocking, 24 hrs/room temperature.
Fig 9.
Supposed mechanism of ammonia and amines interaction with hexachlorofluorescein dye.
R = 5'-ODN-spacer. R' = H (II), tert-Bu (IIa).
Fig 10.
(A) Preparative profile after our debloking method. (B) Analytic HPLC of pure H50 after its re-ammonolysis.
Fig 11.
HPLC profiles of A38B2 after photochemical transformation.
Incubation: (A) 1 hour in conc. NH4OH under the daylight; "18-HQ"-gradient. The structure of diamino-derivative (III) see in Fig 1. (B) - 25 minutes under PCR-mimic conditions, 64°C; HPLC gradient: 8–60% B-buffer / 25 min. Colourless peaks correspond to non-fluorescent derivatives.