Jean Lhomme

Abasic DNA structure, reactivity, and recognition.

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea).

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation.

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

Convenient Preparation of 2-Deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl Chloride

Abstract By using acetyl chloride as HCl generator, the procedure for the Hoffer preparation of the α-chloro sugar 4a was significantly improved. The α-configuration of the chloro atom was confirmed by using NOE measurement. Sequential transformation of 4a to the β-anomer and to the furfuryl derivative 6 was studied.

Synthesis and characterisation of a new DNA-binding bifunctional ruthenium(II) complex

A new DNA-binding molecule, Ru(tap)2POQ2+, in which a polypyridylruthenium(II) complex is linked to an aminochloroquinoline by a flexible chain, has been prepared and characterised (tap = 1,4,5,8-tetraazaphenanthrene and POQ corresponds to a 1,10-phenanthroline linked to an aminochloroquinoline by an aliphatic chain). This complex is regarded as bifunctional because it contains two moieties of different binding modes and photoreactivities vs. DNA. The 1H NMR data of this compound indicate the presence of an equilibrium between two molecular species. The spectroscopic properties of Ru(tap)2POQ2+ in absorption and luminescence are examined and compared with those of the corresponding ruthenium(II) complex which does not contain the aminochloroquinoline moiety: Ru(tap)2phen2+. Luminescence relative quantum yields and lifetimes show that the MLCT excited-state behaviour is influenced by the presence of the linked quinoline. An intramolecular photoinduced electron transfer in one of the two species in equilibrium, is considered to be responsible for a quenching of the ruthenium(II) complex iuminescence. Preliminary results on the binding characteristics of Ru(tap)2POQ2+ to DNA and [poly(d[A-T])]2 from luminescence and thermal denaturation studies are reported. The intramolecular quenching of luminescence in Ru(tap)2POQ2+ is inhibited when it interacts with nucleic acids. Consequently, the resulting emission is more substantially enhanced in the presence of the polynucleotide relative to the luminescence increase observed with the reference complex, Ru(tap)2phen2+.

Synthesis of fluorescent probes for the detection of abasic sites in DNA

Abstract The three luminescent molecules 1–3 were prepared as highly sensitive and specific probes for the quantification of a major DNA lesion, the abasic site. These molecules incorporate in their structure the reactive oxyamino function linked to the Dansyl or Lissamine-Rhodamine fluorophores through amido or polyether chains.

Synthesis of tröger's base analogs derived from 3-aminoacridine and 10-aminobenzo[b][1,7]phenanthroline

Abstract The heterocyclic aromatic amines 10-aminobenzo[b][1,7]phenanthroline 1 and 3-aminoacridine 2 react regioselectively with formaldehyde in acidic medium to yield the Trogers base analogs 7 and 8.

A new fluorescent probe for sensitive detection of carbonyl compounds

A new method for the convenient and sensitive detection of aldehydes and ketones in environmental samples was developed. This method is described in detail for the measurement of carbonyls in water samples, but it is also potentially applicable to their determination in air. Derivatization of carbonyls is performed using N-(5-dimethylamino-1-naphtalenesulphonamido)-3-oxapentane-1,5-dioxyamine (dansyloxyamine, DNSOA). The resulting oxime ethers are separated by using reversed phase liquid chromatography and fluorescence detection. Parameters influencing both reactivity and sensitivity are discussed. The oxyamino function allows efficient chemical derivatization in water without the use of a catalyst. The dansyl fluorophore and the reactive oxyamino group are linked by an etheral chain which leads to very similar fluorescence responses for formaldehyde, acetaldehyde and acetone derivatives. The detection limit of this method compares favourably with classical methods. Furthermore, the analysis procedure is easily set up and can be directly applied to environmental water samples.

Synthesis of Nucleoside Triphosphates that Contain an Aminooxy Function for “Post-Amplification Labelling”

Preparation of the uridine and adenosine triphosphates 1 and 2 bearing a linker with a terminal aminooxy group is described. Both 1 and 2 react readily with the aldehydic fluorescein derivative 15. They could each be incorporated into a 330-mer fragment with T7 RNA polymerase.

The oxyamino-aldehyde coupling reaction: An efficient method for the derivatization of oligonucleotides

Abstract Oligonucleotides containing an aldehydic function at any preselected position in the sequence have been prepared using post-synthetic oxidation of an alkene as the key reaction. These were efficiently coupled to a reporter molecule tethered to an oxyamino linker.

Hydrolysis of 2′-deoxypurine nucleosides. The effect of substitution at the C-8 position

Abstract The hydrolytic stability of 2′-deoxypurine nucleosides is decreased by introduction of electron-withdrawing substituents at the C-8 position in the series of compounds 2–8 , 10–14 . The sulfone group causes a 2.9 × 10 4 rate acceleration for glycosidic bond cleavage in compound 14 .