Chemistry of the 8-Nitroguanine DNA Lesion: Reactivity, Labelling and Repair.

The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source.

SERS and SERRS Detection of the DNA Lesion 8-Nitroguanine: A Self-Labeling Modification.

Rapid and sensitive methods to detect DNA lesions are essential in order to understand their role in carcinogenesis and for potential diagnosis of cancers. The 8-nitroguanine DNA lesion, which is closely associated with inflammation-induced cancers, has been characterized for the first time by surface-enhanced Raman spectroscopy (SERS). This lesion has been studied as the free base, as well as part of a dinucleotide and oligodeoxynucleotides (ODNs) at 5 different excitation wavelengths in the range 785-488 nm. All nitrated samples produced distinctly different spectra from their control guanine counterparts, with nitro bands being assigned by DFT calculations. Additional resonance enhancement was observed at the shorter excitation wavelengths, these SERRS measurements allowed the detection of one nitrated guanine in over 1,300 bases. In addition, SER(R)S can be used to detect whether the unstable lesion is covalently attached to the ODN or has been released by hydrolytic depurination.

Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine.

8-Nitro-2'-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2'-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2'-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase ß (pol ß), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol ß showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson-Crick pair.

Alpha-lithio quinuclidine N-oxide (Li-QNO): a new base for synthetic chemistry.

A-Lithio quinuclidine N-oxide (Li-QNO) behaves as a strong non-nucleophilic base and an HMPA mimetic in a tandem process, in a range of synthetically useful reactions.

Synthesis and transcription studies on 5'-triphosphates derived from 2'-C-branched-uridines: 2'-homouridine-5'-triphosphate is a substrate for T7 RNA polymerase.

The 5'-triphosphates of 2'-hydroxymethyluridine (2'-homouridine) and 2'-hydroxyethyluridine were prepared from the corresponding acetyl-protected nucleosides by initial phosphitylation with 2-chloro-5,6-benzo-1,2,3-dioxaphosphorin-4-one. 2'-Acetamidouridine 5'-triphosphate was prepared in an analogous fashion from uridine 2'-C-, 3'-O-gamma-butyrolactone, in which the 3'-hydroxyl group is internally protected as the lactone. Subsequent treatment with ammonia gave the required acetamido triphosphate. All three triphosphates were investigated as substrates for T7 RNA polymerase and a Y639F mutant of this enzyme. 2'-Homouridine triphosphate was found to be a substrate for the wild-type enzyme in the presence of manganese and was specifically incorporated into short RNA transcripts (20 and 21 nucleotides in length). The presence of the analogue within the transcripts was confirmed through its resistance to alkaline hydrolysis. Gel electrophoretic analysis also showed that 2'-homouridine could be multiply incorporated into a transcript with a length of 75 nucleotides. This is the first report of a 2'-deoxy-2'-alpha-C-branched nucleoside 5'-triphosphate acting as a substrate for T7 RNA polymerase. The 2'-hydroxyethyl- and 2'-acetamido -uridine triphosphates were not substrates for the enzymes.

Synthesis of phosphorothioamidites derived from 3'-thio-3'-deoxythymidine and 3'-thio-2',3'-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.

Isoquine and related amodiaquine analogues: a new generation of improved 4-aminoquinoline antimalarials.

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects including agranulocytosis and liver damage. The observed drug toxicity is believed to involve the formation of an electrophilic metabolite, amodiaquine quinoneimine (AQQI), which can bind to cellular macromolecules and initiate hypersensitivity reactions. We proposed that interchange of the 3' hydroxyl and the 4' Mannich side-chain function of amodiaquine would provide a new series of analogues that cannot form toxic quinoneimine metabolites via cytochrome P450-mediated metabolism. By a simple two-step procedure, 10 isomeric amodiaquine analogues were prepared and subsequently examined against the chloroquine resistant K1 and sensitive HB3 strains of Plasmodium falciparum in vitro. Several analogues displayed potent antimalarial activity against both strains. On the basis of the results of in vitro testing, isoquine (ISQ1 (3a)) (IC(50) = 6.01 nM +/- 8.0 versus K1 strain), the direct isomer of amodiaquine, was selected for in vivo antimalarial assessment. The potent in vitro antimalarial activity of isoquine was translated into excellent oral in vivo ED(50) activity of 1.6 and 3.7 mg/kg against the P. yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine. Subsequent metabolism studies in the rat model demonstrated that isoquine does not undergo in vivo bioactivation, as evidenced by the complete lack of glutathione metabolites in bile. In sharp contrast to amodiaquine, isoquine (and Phase I metabolites) undergoes clearance by Phase II glucuronidation. On the basis of these promising initial studies, isoquine (ISQ1 (3a)) represents a new second generation lead worthy of further investigation as a cost-effective and potentially safer alternative to amodiaquine.

Studies on 2'-alpha-C-carboxyalkyl nucleosides and their application to a stereocontrolled nucleobase exchange process.

The ability of 2'-alpha-C-carboxyalkyl nucleosides to undergo an unusual two-step stereocontrolled nucleobase exchange process has been investigated. Upon silylation a protected 2'-deoxy-2'-alpha-C-(carboxymethyl)uridine derivative can undergo intramolecular displacement of the uracil base, by the 2'-carboxylic acid group, to form a pentofuranosyl gamma-lactone. Under identical conditions the homologous 2'-deoxy-2'-alpha-C-(carboxyethyl)uridine derivative does not yield the corresponding delta-lactone, but undergoes elimination of uracil to give the corresponding glycal. The pentofuranosyl gamma-lactone is a good substrate for nucleoside synthesis by the Vorbrüggen procedures and undergoes completely stereoselective ring opening with either pyrimidine or purine silylated nucleobases to give novel 2'-C-carboxymethyl beta-nucleosides in moderate to high yield.

Probing the effect of a 3'-S-phosphorothiolate link on the conformation of a DNA:RNA hybrid; implications for antisense drug design.

The effects of a single 3'-S-phosphorothiolate link in the DNA strand of a DNA:RNA dodecamer duplex is described; the sulfur induces a conformational shift in the (attached) sugar pucker, as shown by 1H NMR studies, and increases the thermal stability of the duplex compared to the non-modified system.