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-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer's disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

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.

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. A comprehensive study of the two possible tautomeric forms, through a detailed characterization of this compound, has implications for its base pairing properties. The target compound was obtained through a synthetic sequence of five steps, where all intermediates were fully characterized using spectral data. The analysis of the two tautomers was then evaluated through NMR spectroscopy and theoretical calculations of the chemical shifts and NH coupling constants, which were also compared with the data from guanosine.

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. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

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. One outstanding question regarding the mechanistic model for SgrAI is whether or not the activating primary site DNA must be cleaved by SgrAI prior to inducing activation. Herein we show that an uncleavable primary site DNA containing a 3'-S-phosphorothiolate is in fact able to induce activation. In addition, we now show that cleavage of secondary site DNA can be activated to nearly the same degree as primary, provided a sufficient number of flanking base pairs are present. We also show differences in activation and cleavage of the two types of secondary site, and that effects of selected single site substitutions in SgrAI, as well as measured collisional cross-sections from previous work, are consistent with the cryo-electron microscopy model for the run-on activated oligomer of SgrAI bound to DNA.

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. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3'-S-phosphorothiolate (3'-SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. (1) H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3'-SP modifications is more compact. The 3'-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures.

Thermal stabilisation of RNA·RNA duplexes and G-quadruplexes by phosphorothiolate linkages.

The effect of 3'-S-phosphorothiolate linkages on the stability of RNA·RNA duplexes and G-quadruplex structures has been studied. 3'-Thio-2'-deoxyuridine was incorporated into RNA duplexes and thermal melting studies revealed that the resulting 3'-S-phosphorothiolate linkages increased the stability of the duplex to thermal denaturation. Additionally, and contrary to expectation, a similar effect on duplex stability was observed when the same thionucleoside was incorporated into the RNA strand of a RNA·DNA duplex. A suitably protected derivative of 3'-thio-2'-deoxyguanosine was prepared using an oxidation-reduction strategy and this residue also increased the thermal stability the [d(TGGGGT)](4) G-quadruplex when positioned centrally. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and imply that the previously noted high thermal stability of parallel RNA quadruplexes is not derived from H-bonding interactions of the 2'-hydroxyl group, but can be attributed to conformational effects.

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.

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. Full experimental details and spectroscopic data for the synthetic intermediates and the target dinucleotide are provided.

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. Key to the RNAi pathway are the small-interfering RNAs (siRNAs), which at 21-23 nucleotides in length are very amenable to analogue development by chemists. However in comparison to the use of oligonucleotides as antisense agents, an area where many chemists first developed an interest in nucleic acids, the RNAi pathway is exceedingly complex. The literature is also complicated by the fact that the phenomenon has been studied in a wide range of organisms. In this tutorial review we have presented the subject from a more chemical perspective, incorporating a glossary to give a clear explanation of the specialist terms. However, the coverage of the biology remains sufficiently detailed to give the reader the necessary insight that we believe will be essential for the successful design of chemically modified siRNA.

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. 5'-->3' Synthesis of oligonucleotides gave relatively poor yields for the internal incorporation of 3'-thionucleosides [to give a 3'-S-phosphorothiolate (3'-SP) linkage] and multiple 3'-SP modifications could not be introduced by this method. However, the reverse direction approach provided an efficient route to oligonucleotides terminating with a 3'-thionucleoside. The direct synthesis of these thio-terminating oligomers has not previously been reported and the methods described are applicable to 2'-deoxy-3'-thionucleosides derived from thymine, cytosine and adenine.

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. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.

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.

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.

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.

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. Significantly, success of the coupling reactions could be monitored by NMR without loss of materials or termination of the synthetic sequence and this promises much for the future of both oligonucleotide and polypeptide synthesis.

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.