Purine bases modification

The conformational changes in DNA induced by aromatic amine-purine base modifications are important determinants of the adduct s biological activity and... 

The analogs of pyrimidine and purine bases can be derived by purely formal structural modifications or, more rationally, from the results of biochemical investigation. 

One of the most important reactions of purines is the bromination of guanine or adenine at the C-8 position. It is this site that is the most common point of modification for bioconjugate techniques using purine bases (Figure 1.53). Either an aqueous solution of bromine or the compound N-bromosuccinimide can be used for this reaction. The brominated derivatives then can be used to couple amine-containing compounds to the pyrimidine ring structure by nucleophilic substitution (Chapter 27, Section 2.1). 

Of the purine nucleosides, dATP may be derivatized at its N-6 position using a long linker arm terminating in a detectable group without losing the ability to be enzymatically incorporated into DNA probes. By contrast, if modification is done at the C-8 position of purine bases, DNA polymerase cannot by used to add the labeled monomer to an existing strand. C-8 derivatives, however, can be added at the 3 terminal using terminal transferase enzyme. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Our inhibitor design strategy was based on the premise that structural modifications in the base of purine riboside that enhance purine base hydration without impairing the binding of the hydrated species to the ADA binding site would result in purine riboside (PR) analogues with high ADA inhibitory potency. Since the apparent inhibition constant (Kj (app)) is related to the hydration equilibrium constant (Keq) and the inhibitory constant for the hydrated molecule (Kj ) by... 

Figure 3. Purine riboside (PR) hydration and the effect of base modification Y.

Next, some typical examples will be presented of how a DNA-electrochemical biosensor is appropriate to investigate the DNA damage caused by different types of substances, such as the antioxidant agent quercetin (Scheme 20.1), an anticancer drug adriamycin (Scheme 20.2) and nitric oxide. In all cases, the dsDNA damage is detected by changes in the electrochemical behaviour of the immobilized dsDNA, specifically through modifications of the purinic base oxidation peak current [3,5,40]. 

Novel congeners of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 32), the dialkyl esters of purine and pyrimidine A-[2-(phosphonomethoxy)ethyl] derivatives substituted at position 2, 6 or 8 of the purine base (33) or position 2, 4 or 5 of the pyrimidine base (34), were prepared by alkylation of the appropriate heterocyclic base with 2-chloroethoxymethylphosphonate diester. Except for the 5-bromo-cytosine derivative (34a), no activity against DNA viruses or retroviruses was observed for the novel pyrimidine analogues. However, modifications to the purine led to compounds highly active against HSV-1 and -2, VZV, CMV, W, MSV and HIV. 

The base modifications discussed so far are mostly concerned with those permissible in the pyrimidine motif (Y RY). In the purine motif (R RY), the permitted triads are A-AT, T AT and G GC, and the following examples illustrate the permissible base modifications in this motif. 

Some potent photooxidants can also produce outer-sphere electron transfer from the DNA. Here it is the guanine bases, likely those stacked with neighboring purines, that are most easily oxidized and hence most susceptible to attack. Again, this base modification requires alkaline treatment to convert the... 

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. 

Tendency towards triple helix formation strongly depends on nucleotide structures. Thus, a decrease in tendency is observed with a change in the sugar moiety with deoxyribose < ribose < 2 -0-methylribose [249], With phosphorothioates, triple helix formation depends strongly on the sequence. A modification next to the 5 -position in presence of a purine base increases stability of the triple helix, while a modification next to the 5 -position in presence of a pyrimidine base decreases stability [250],... 

Base modification at the 4-position of pyrimidines leads to loss of base-pairing properties. The thiol moiety in 91 was deprotected after ODN synthesis using 1 M DBU in acetonitrile. The modified oligothymidylate was then cleaved from the solid phase and reacted with N-(2-chloroethylthio)phthalimide to yield 92, which was subjected to further derivatization [264]. Purine base positions accessible for ligand attachment are C-8 of adenosine (93) [265] and C-2 of guanosine (94) [266]. 

The pyrimidine bases cytosine, uracil, and thymine cannot be directly alkylated by halo sugars. The nucleophilicity of the pyridone-type ring nitrogens is much reduced relative to the pyridine-type nitrogens present in the purine bases. The dialkoxypyrimidines are reactive, and dealkylation occurs at the alkylated nitrogen. This is known as the Hilbert-Johnson procedure A recent modification employs... 

In table II I the Influence of base modifications in the purine(P)ribosides upon the conformations preferred by... 

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