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International audience; Alkylation of zinc-bound thiolates occurs in both catalytic and structural zinc sites of enzymes. Recent biomimetic studies have led to a controversy as to which mechanism is operative in thiolate alkylation. Building on one of these biomimetic complexes, we have devised a series of models that allow for an appraisal of the roles of charge, ligand nature, and hydrogen bonding to sulfur on reactivity. The reactions of these complexes with methyl iodide, leading to thioethers and zinc iodide complexes, have been examined by density functional theory calculations, in the gas phase as well as in an aqueous solution. In all cases, a S(N)2 reaction is favored over sigma-bond metathesis. Both the net electronic charge and the hydrogen bond play a significant role in the nucleophilicity of the thiolate. We find that the mechanistic diversity observed experimentally can be explained by the difference in the net charge of the complexes. A dianionic complex follows a dissociative pathway, whereas an associative one is preferred for a neutral system.

International audience; The proton affinities of a series of mono- and polynuclear zinc-thiolate compounds, mimicking zing finger protein and metallothioneins sites, have been investigated by Density Functional Theory calculations. This allows to evaluate the intrinsic nucleophilicity of synthetic and natural zinc-bound thiolates and to compare their relative reactivities. The site specificity of the experimental thiolate alkylation for the Zn4Cys11 clusters in metallothioneins is well reproduced, as well as the relative reactivities of ZnHis2Cys2, ZnHisCys3 and ZnCys4 zinc finger sites. Our results also show that terminal thiolates, bound to only one Lewis acid, are more reactive than bridging thiolates. Synthetic inorganic clusters and the Zn4Cys9His2 cluster found in a cyanobacterial metallothionein are less reactive than Zn4Cys11 and Zn3Cys9 clusters in metallothioneins. These results allow discussing the influence of the protein backbone and residues on the reactivity of these natural clusters.

Density Functional Theory and post-Hartree Fock calculations reveal an unusual energy profile for Zn-S and Zn-N bond dissociation reactions in several [Zn(SR)(4)](2-) and [Zn(Im)(SR)(3)](-) complexes. The Zn-S bond dissociation in tetrathiolate dianions, which is highly exothermic in the gas phase, proceeds through a late transition state which can be rationalized on the basis of an avoided crossing resulting from Coulomb repulsion between the anionic fragments and ligand-to-metal charge-transfer in the [Zn(SR)(4)](2-) complexes. When solvation models for water, DMSO, or acetonitrile are included, some complexes become stable while others are metastable, so this constitutes the first theoretical model which is in full agreement with the experimental data for various [Zn(SR)(4)](2-), [Zn(SR)(3)](-), and [Zn(Im)(SR)(3)](-) complexes. The analysis given here indicates that the Zn(Cys)(4) and Zn(His)(Cys)(3) cores of numerous proteins are metastable with respect to Zn-S and Zn-N bond dissociation, respectively. This is consistent with the kinetic lability at the zinc-centers and illustrates that in nature, thermodynamic stability is imparted upon the zinc cores by the protein environment.

International audience; The nucleophilicity of biomimetic zinc-thiolate complexes against methyl iodide has been studied. The activation barrier has been computed with the B3LYP functional. The reactivity depends on the global charge, the nature of the ligand set and the presence of hydrogen bonds. This shows that the understanding of zinc site nucleophilicity cannot be achieved by the knowledge of the atom donor set for zinc only, as currently done in the literature. Moreover, we show that sulfur proton affinity and activation barrier are directly proportional, thus providing a good nucleophilicity index for zinc-bound thiolate.

BBG2Na, a well-defined recombinant protein produced in Escherichia coli, is a promising human respiratory syncytial virus subunit vaccine candidate. This study describes the quantification of residual DNA in large scale batches used in phase I to III clinical trials. Two different analytical methods were developed and applied on five different final bulks of Drug Substance and their associated in process control samples, namely a chemiluminescent hybridisation assay and the total DNA Threshold System assay. These two complementary methods demonstrated the clearance of residual DNA during the downstream purification process. The amount of residual DNA found in the final bulks was below 20 pg of DNA per 300 microg BBG2Na, the highest tested clinical dose of antigen. This is very low level of residual DNA for a recombinant subunit vaccine produced in a bacteria and contribute to make for BBG2Na a well-characterised biopharmaceutical. This study also provides data concerning the validation of the hybridisation dot blot assay and the total DNA Threshold(trade mark)assay.