Structure-based design of nucleoside-derived analogues as sulfotransferase inhibitors.

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group ('sulfation') from the enzyme co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2--sulfotransferase (HS2ST1).

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.

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.

Tautomerism in 8-Nitroguanosine Studied by NMR and Theoretical Calculations.

The guanine base in DNA, due to its low oxidation potential, is particularly sensitive to chemical modifications. A large number of guanine lesions have been characterized and studied in some detail due to their relationship with tissue inflammations. Nevertheless, one example of these lesions is the formation of 8-nitro-guanosine, but the NMR data of this compound was only partially interpreted.

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site.

Probing the run-on oligomer of activated SgrAI bound to DNA.

SgrAI is a type II restriction endonuclease with an unusual mechanism of activation involving run-on oligomerization. The run-on oligomer is formed from complexes of SgrAI bound to DNA containing its 8 bp primary recognition sequence (uncleaved or cleaved), and also binds (and thereby activates for DNA cleavage) complexes of SgrAI bound to secondary site DNA sequences which contain a single base substitution in either the 1st/8th or the 2nd/7th position of the primary recognition sequence. This modulation of enzyme activity via run-on oligomerization is a newly appreciated phenomenon that has been shown for a small but increasing number of enzymes.

Stabilization of a Bimolecular Triplex by 3'-S-Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation.

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single-stranded nucleic acids are also relevant in third-strand binding.

Artemisinin-polypyrrole conjugates: synthesis, DNA binding studies and preliminary antiproliferative evaluation.

Greater than the sum of its parts: Artemisinins are currently in phase I-II clinical trials against breast, colorectal and non-small-cell lung cancers. In an attempt to offer increased specificity, a series of hybrid artemisinin-polypyrrole minor groove binder conjugates are described. DNA binding/modelling studies and preliminary biological evaluation give insights into their mechanism of action and the potential of this strategy.

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.

Dinucleotides containing 3'-S-phosphorothiolate linkages.

The 3'-S-phosphorothiolate (3'-SP) linkage has proven to be a very useful analogue of the phosphodiester group in nucleic acid derivatives; it is achiral and also shows good resistance to nucleases. Whilst oligonucleotides containing a 3'-SP linkage are best prepared using phosphoramidite chemistry, the corresponding dinucleotides are most efficiently synthesised using a Michaelis-Arbuzov reaction between a nucleoside 5'-phosphite and a nucleoside 3'-S-disulphide. The method described here is for a thymidine dinucleotide and is based on the use of a silyl phosphite, which is more reactive than simple alkyl phosphites and also simplifies the deprotection strategy.

RNA interference: a chemist's perspective.

Since the first unequivocal description of RNA interference (RNAi) in 1998, it has remained one of the hottest topics under investigation, culminating in the award of a Nobel Prize to its discoverers in 2006. Excitement over this technique derives from the ease with which it can be used to switch-off a specific gene in almost any organism, thereby allowing the role of that gene to be identified. More importantly, it offers the potential to treat certain diseases by switching-off the causative genes.

Reverse-direction (5'-->3') synthesis of oligonucleotides containing a 3'-S-phosphorothiolate linkage and 3'-terminal 3'-thionucleosides.

The synthesis of oligodeoxynucleotides containing 3'-thionucleosides has been explored using a reverse-direction (5'-->3') approach, based on nucleoside monomers which contain a trityl- or dimethoxytrityl-protected 3'-thiol and a 5'-O-phosphoramidite. These monomers are relatively simple to prepare as trityl-based protecting groups were introduced selectively at a 3'-thiol in preference to a 5'-hydroxyl group. As an alternative approach, trityl group migration could be induced from the 5'-oxygen to the 3'-thiol function.

A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site.

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands.

Antitumour and antimalarial activity of artemisinin-acridine hybrids.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells.

A series of artemisinin dimers incorporating a metabolically stable C-10 carba-linkage have been prepared, several of which show remarkable in vitro antimalarial activity (as low as 30 pM) versus Plasmodium falciparum and in vitro anticancer activity in the micromolar to nanomolar range versus HL-60 cell lines.

Towards fluorous-phase synthesis of nucleoside beta-peptides.

We have recently shown that peptides derived from nucleoside beta-amino acids adopt an unusual 8-helical conformation in solution(1). These beta-peptide homooligomers were constructed using Fmoc solid-phase peptide synthesis protocols (SPPS); however, we found these procedures to be somewhat limiting in terms of scale and lacking a quantitative method of monitoring reactions. Preliminary investigations into the use of fluorous dendrons as an alternative to SPPS have been conducted and show that coupling reactions with modified nucleoside (1) and fluorous dendrimer (2) are possible in the presence of a glycine linker.

Incorporation of 3'-S-phosphorothiolates into RNA: potential applications in RNAi.

Using solid-phase synthesis, oligoribonucleotides containing multiple 3'-S-phosphorothiolate modifications have been successfully synthesized, purified and characterized, utilizing a 2'-deoxyuridine phosphorothioamidite monomer.

Foldamers derived from nucleoside beta-amino acids: a new twist on the DNA helix.

Peptides derived from a thymidine beta-amino acid have been prepared by solid-phase synthesis and their conformation investigated by NMR. Interestingly, NMR and modelling studies indicate that the tetramer and octamer form an unusual 8-helical conformation. Studies are currently underway to investigate the synthesis of peptides derived from the other deoxyribonucleosides with the intention of examining the association between helices capable of nucleobase-pairing.

The effect of 2'-fluorine substitutions on DNA i-motif conformation and stability.

(1)H and (19)F NMR, and UV thermal melting studies have established that the stability of d(TCCCCC) is enhanced by the inclusion of a single 2'-fluorine-modified deoxycytidine residue; the results support the notion of the importance of sugar-sugar contacts in stabilising i-motifs in general and reveal that solvation is the cause of the instability of RNA equivalents.

Peptides derived from nucleoside beta-amino acids form an unusual 8-helix.

Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

Synthesis of the 3'-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

Oligodeoxynucleotides containing 3'-S-phosphorothiolate (3'-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2'-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3'-PS linkages. The synthesis of the 5'-protected-3'-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.

Synthesis of 3'- S-phosphorothiolate oligonucleotides for their potential use in RNA interference.

The potency of RNA interference (RNAi) undoubtedly can be improved through chemical modifications to the small interfering RNAs (siRNA). By incorporation of the 3'-S-phosphorothiolate modification into strands of RNA, it is hoped that specific regions of a siRNA duplex can be stabilised to enhance the target binding affinity of a selected antisense strand into the activated RNA-induced silencing complex (RISC*). Oligonucleotides composed entirely of this modification are desirable so unconventional 5' --> 3' synthesis is investigated, with initial solution-phase testing proving successful.

Foldamers derived from nucleoside beta-amino acids: PNA Or DNA? Can we have both in one place?

The synthesis of a modified thymidine (nucleoside beta-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.

Stabilization of multi-stranded nucleic acid structures using 3'-S-phosphorothiolate linkages.

3 '-S-Phosphorothiolate linkages incorporated into an oligodeoxynucleotide have been shown to stabilise duplex formation with a complementary RNA strand, but destabilise a duplex formed with a complementary DNA strand. The four-stranded i-motif structure is also stabilised this modification.