Purine bases, fluorescence

Diaminobenzoic acid (DABA) reacts with aldehydes of the form RCH CHO to produce a strongly fluorescent compound. Acid-catalysed removal of the purine base from the nucleic acid exposes the 1 and 2 carbons of deoxyribose... 

The obvious similarity between the purine bases of DNA and pteridines, especially between guanosine and pterins, has encouraged extensive studies of the synthesis and properties of pteridine-containing nucleoside and nucleotides. Synthetic methods have naturally built upon established methods of nucleic acid synthesis. The primary property of use in applications of these compounds to DNA chemistry is fluorescence, which is very much greater for pteridines than for purines. 

The natural purine bases adenine and guanine do not show appreciable fluorescence under neutral conditions at room temperature. It was also found that purine itself shows only very low fluorescence intensity as a neutral molecule, but this increases markedly on either acid or alkaline titration. For the purine monocation, emission at 400 nm with a quantum yield (4>f) of 0.008 was observed for the anion emission at 370 nm with of 0.045 was found. Appreciable fluorescence at room temperature can be observed for the adenine cation (Table 16), while, similarly, guanine in neutral solution is also nonfluorescent but emission becomes appreciable on protonation. However, with an increase of the pH beyond 11, decreased fluorescence is observed. A case where the neutral molecule is more fluorescent than either the cation or the anion is presented by purin-2-amine or purine-2,6-diamine as well as their nucleosides (Table 16, 5-7). 

Deprotection involving concentrated ammonia is a critical step in the synthetic cycle. In particular, the use of (fluorescent) dyes linked to oligonucleotides or some other specific modifications is limited due to their lack of stability in the presence of concentrated ammonia solution, which is required for cleavage of the amides. Sometimes an improvement in deprotection yields may be achieved by an increase of reaction temperature from 55°C to 80°C, which reduces reaction time to 60 min [48]. A prerequisite for significantly milder cleavage protocols, however, is a change in protective groups at pyrimidine and purine bases. 

The fluorescence quantum yields for all NABs are very low in aqueous solutions at room temperature, the excitation energy being dissipated through the nonradiative decay channels. On the other hand protonated purines show fluoresc ice at room temperature as well as after being absorbed on a chromatographic p >er. To obtain insight into the excited state properties of the neutral bases and nucleotides in polar solvents, different low temperature experiments were performed. The first low temperature work on nucleic acids was reported in I960, while the phosphorescence of nucleic acids was first reported for adenine derivatives in 1957. The first... 

Three fluorescent purine analogs will be described here 2-aminopurine (2AP) [183] and 8-vinyladenine (8VAD), both of which mimic adenine, and N1,N6-ethenoadenine (e-AD). Their structures are shown in Figure 11-15. 2AP is probably the most widely used fluorescent DNA base analog, being utilized as a probe for DNA conformational dynamics [184, 185], due to the environmental specificity of... 

Daniels M, Hauswirth W (1971) Fluorescence of the purine and pyrimidine bases of the nucleic acids in neutral aqueous solution at 300 K. Science 171 675... 

The close correspondence of the DNA absorption spectrum with that of a mixture of mononucleotides of the same composition illustrates the weak nature of the interactions between neighboring purine and pyrimidine bases guanine (G), cytosine (C), adenine (A), and thymine (T) at an interplanar separation of 3.36 A in the unexcited double-helical configuration. On the other hand the structureless fluorescence band of (calf-thymus) DNA is red-shifted by 3500 cm-1 from the fluorescence spectral origin of the mononucleotides it closely resembles the fluorescence spectrum of the dinocleotide ApT (and of poly dAT) and is accordingly identified131 with the fluorescence... 

Fluorescence assays are considered among the most convenient, sensitive, and versatile of all laboratory techniques. However, the purine and pyrimidine bases yield only weak fluorescence spectra. Le Pecq and Paoletti (1967) showed that the fluorescence of a dye, ethidium bromide, is enhanced about 25-fold when it interacts with DNA. Ethidium bromide, which is a relatively small planar molecule (Figure El3.4), binds to DNA by insertion between stacked base pairs (intercalation). The process of intercalation is especially significant for aromatic dyes, antibiotics, and other drugs. Some dyes, when intercalated into DNA, show an enhanced fluorescence that can be used to detect DNA molecules after gel electrophoresis measurements (see Chapter 4 and Experiments 14 and 15) and to characterize the physical structure of DNA. Two analyses of DNA will be completed in this experiment ... 

Another promising approach to 2 -deoxyribonucleosides with only a slightly modified purine which shows selective base pairing with 2 -deoxythymidinc (dT) and can be incorporated into DNA is represented by compounds of type 10. Here, position 7 of a 7-deazapurine heterocycle is substituted by an alkynyl side chain. Such nucleosides, e.g. 7-deaza-2 -deoxy-7-hex-l-ynyl-adenosine, exhibit strong fluorescence = 275 nm, = 405 nm). ... 

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