Acetohydroxamic acid salts: mild, simple and effective degradation reagents to counter Novichok nerve agents.

Novichoks is the latest known class of organophosphorus nerve agents to be developed. These highly lethal persistent agents, which exert their toxicity mainly through dermal exposure, pose new major challenges in mitigating their effect, mainly in respect to decontamination and medical countermeasures. Herein we report on the effective degradation of Novichok agents (A-230, A-232 and A-234) by hydroxamic acid salts. This class of α-nucleophiles, with emphasis on the FDA approved drug acetohydroxamic acid, were found to promote rapid hydrolysis of these extremely toxic agents. Using 31P NMR the Novichoks degradation rates were determined to be in time scale of minutes with the following order of reactivity A-230>A-232>A-234. The degradation efficiency was found to be dependent on the nucleophiles, their counter-cations and the specific solvent mixture used. Hence, these scavengers can serve as efficient and mild decontaminants in various scenarios including surfaces, dermal decontamination (as an alternative to active lotions such as the RSDL® kit) and also as a medical countermeasure in the form of "catch-up therapy".

Highly sensitive chemiluminescence sensors for the detection and differentiation of chemical warfare agents.

Highly sensitive chemiluminescence-based probes that effectively detect and differentiate between the extremely toxic real G- and V-type organophosphorus chemical warfare agents (OPCWAs) are presented. This straightforward approach does not require any instrumentation or light source; hence, it appears ideal for the future development of field colorimetric detectors.

Placing CF2 in the Center: Major Physicochemical Changes Upon a Minor Structural Alteration in Gem-Difunctional Compounds.

Fluorine atoms play an important role in all branches of chemistry and accordingly, it is very important to study their unique and varied effects systematically, in particular, the structure-physicochemical properties relationship. The present study describes exceptional physicochemical effects resulting from a H/F exchange at the methylene bridge of gem-difunctional compounds. The Δlog P(CF2-CH2) values, that is, the change in lipophilicity, observed for the CH2 /CF2 replacement in various α,α-phenoxy- and thiophenoxy-esters/amides, diketones, benzodioxoles and more, fall in the range of 0.6-1.4 units, which for most cases, is far above the values expected for such a replacement. Moreover, for compounds holding more than one such gem-difunctional moiety, the effect is nearly additive, so one can switch from a hydrophilic compound to a lipophilic one in a limited number of H/F exchanges. DFT studies of some of these systems revealed that polarity, conformational preference as well as charge distributions are strongly affected by such hydrogen to fluorine atom substitution. The pronounced effects described, are a result of the interplay between changes in polarity, H-bond basicity and molecular volume, which were obtained with a very low 'cost' in terms of molecular weight or steric effects and may have a great potential for implementation in various fields of chemical sciences.

Studying Lipophilicity Trends of Phosphorus Compounds by 31P-NMR Spectroscopy: A Powerful Tool for the Design of P-Containing Drugs.

Systematically studying the lipophilicity of phosphorus compounds is of great importance for many chemical and biological fields and particularly for medicinal chemistry. Here, we report on the study of trends in the lipophilicity of a wide set of phosphorus compounds relevant to drug design including phosphates, thiophosphates, phosphonates, thiophosphonates, bis-phosphonates, and phosphine chalcogenides. This was enabled by the development of a straightforward log P determination method for phosphorus compounds based on 31P-NMR spectroscopy. The log P values measured ranged between -3.2 and 3.6, and the trends observed were interpreted using a DFT study of the dipole moments and by H-bond basicity (pKHB) measurements of selected compounds. Clear signal separation in 31P-NMR spectroscopy grants the method high tolerability to impurities. Moreover, the wide range of chemical shifts for the phosphorus nucleus (250 to -250 ppm) enables a direct simultaneous log P determination of phosphorus compound mixtures in a single shake-flask experiment and 31P-NMR analysis.

Structural Studies Reveal the Role of Helix 68 in the Elongation Step of Protein Biosynthesis.

The ribosome, a multicomponent assembly consisting of RNA and proteins, is a pivotal macromolecular machine that translates the genetic code into proteins. The large ribosomal subunit rRNA helix 68 (H68) is a key element in the protein synthesis process, as it coordinates the coupled movements of the actors involved in translocation, including the tRNAs and L1 stalk. Examination of cryo-electron microscopy (cryo-EM) structures of ribosomes incubated for various time durations at physiological temperatures led to the identification of functionally relevant H68 movements. These movements assist the transition of the L1 stalk between its open and closed states. H68 spatial flexibility and its significance to the protein synthesis process were confirmed through its effective targeting with antisense PNA oligomers. Our results suggest that H68 is actively involved in ribosome movements that are central to the elongation process. IMPORTANCE The mechanism that regulates the translocation step in ribosomes during protein synthesis is not fully understood. In this work, cryo-EM techniques used to image ribosomes from Staphylococcus aureus after incubation at physiological temperature allowed the identification of a conformation of the helix 68 that has never been observed so far. We then propose a mechanism in which such helix, switching between two different conformations, actively coordinates the translocation step, shedding light on the dynamics of ribosomal components. In addition, the relevance of helix 68 to ribosome function and its potential as an antibiotic target was proved by inhibiting Staphylococcus aureus ribosomes activity in vitro using oligomers with sequence complementarity.

Origin of life: protoribosome forms peptide bonds and links RNA and protein dominated worlds.

Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.

Modulation of the H-Bond Basicity of Functional Groups by α-Fluorine-Containing Functions and its Implications for Lipophilicity and Bioisosterism.

Modulation of the H-bond basicity (pKHB) of various functional groups (FGs) by attaching fluorine functions and its impact on lipophilicity and bioisosterism considerations are described. In general, H/F replacement at the α-position to H-bond acceptors leads to a decrease of the pKHB value, resulting, in many cases, in a dramatic increase in the compounds' lipophilicity (log Po/w). In the case of α-CF2H, we found that these properties may also be affected by intramolecular H-bonds between CF2H and the FG. A computational study of ketone and sulfone series revealed that α-fluorination can significantly affect overall polarity, charge distribution, and conformational preference. The unique case of α-di- and trifluoromethyl ketones, which exist in octanol/water phases as ketone, hemiketal, and gem-diol forms, in equilibrium, prevents direct log Po/w determination by conventional methods, and therefore, the specific log Po/w values of these species were determined directly, for the first time, using Linclau's 19F NMR-based method.

Active and Strippable PVA/Borax/NaBO3 Hydrogel for Effective Containment and Decontamination of Chemical Warfare Agents.

Active gels present unique potential for the decontamination of chemical warfare agents (CWAs) as they strongly adhere to surfaces, thus allowing prolonged decontamination time. Herein, we present a decontamination hydrogel based on polyvinyl alcohol/borax, which contains sodium perborate (NaBO3), as an in situ source of the active ingredient hydrogen peroxide. Developed as a binary formulation, this gel instantly forms and effectively sticks when sprayed on various matrices, including porous and vertically positioned matrices. The gel efficiently detoxified the CWAs sarin (GB), O-ethyl S-2-(diisopropylamino)ethyl methylphosphonothioate (VX), and sulfur mustard (HD) in test tubes (2 µL CWA/0.5 mL gel) to provide nontoxic products with reaction half-lives of <3, 45 and 113 min, respectively. The gel was also shown to efficiently decontaminate surfaces contaminated with VX (5-7 mg, 8-12 mL of gel, i.e., >99%) and to prevent GB evaporation, as proven by laboratory wind tunnel experiments. The universal decontamination abilities of this mild hydrogel, as well as its facile application and removal processes suggest that it holds high potential for future development as a new CWA decontamination tool.

Effective neutralization of chemical warfare agents (HD, VX) by Me-DABCOF: a small molecule with dual action.

The ability of mono N-methyl-1,4-diazabicyclo[2.2.2]octane fluoride (Me-DABCOF, 1) to act as a bifunctional reagent that effectively and universally neutralizes both the persistent and extremely toxic blister agent HD and the nerve agent VX in nearly neutral aqueous solution, alumina powder or a hydrogel formulation, is described.

Degradation of Sulfur Mustard on KF/Al2O3: The Role of Organic Solvents and Active Species.

Solvent effects on the ability of KF/Al2O3 supports to degrade the warfare agent sulfur mustard (HD) were explored. RP-KF/Al2O3 possessing hydroxide ions and ECUF/KF/Al2O3 holding fluoride ions were examined. Reactions on RP-KF/Al2O3 containing 10 wt % of organic solvents were faster than those on ECUF/KF/Al2O3. Additionally, RP-KF/Al2O3 led to elimination products, while ECUF/KF/Al2O3 mainly led to substitution derivatives. Enlarging the solvent amounts to 90 wt % resulted in decreased reaction rates. The significance of solvent identity/amount and active species is discussed.

Solvent Effects on the Reactions of the Nerve Agent VX with KF/Al2O3: Heterogeneous or Homogeneous Decontamination?

Solvent effects on the reactions of the extremely toxic nerve agent VX with KF/Al2O3 powder were explored. Small quantities of water or methanol (5-10 wt %), which effectively mobilized all components while maintaining the heterogeneous nature of the reaction, promoted much faster rates than those obtained with larger quantities. Any amount of acetonitrile resulted in extremely slow transformations. Surprisingly, 5-50 wt % of heptane led to fast reactions due to the combination of its ability to mediate fast diffusion of VX and a MAS centrifugation effect.

Component mobility by a minute quantity of the appropriate solvent as a principal motif in the acceleration of solid-supported reactions.

The effects solvents have on fluoride-promoted heterogeneous hydrolysis and alcoholysis of various organo-phosphorus (OP) compounds on the surface of KF/Al2O3 are described. Solid-state magic angle spinning NMR analyses and SEM microscopy have shown that not only is the identity of the solvent important in these reactions but also its quantity. That is, minimal solvent amounts are favored and much more effective in such solid-supported reactions (and maybe generally) than those featuring solvent-free or excess solvent (>50 wt %) conditions. The addition of a minute quantity of the correct solvent (3-10 wt %, molar equivalent scale) avoids reagents leaching from the matrix, permits mobility (mass transport) of the reaction components and ensures their very high local concentration in close proximity to the solid-support large porous surface area. Accordingly, significant acceleration of reactions rates by orders of magnitude is obtained. Fascinatingly, even challenging phosphoesters with poor leaving groups, which were found to be very stable in the presence of solvent-free KF/Al2O3 or wetted with excess water, were efficiently hydrolyzed with a minute amount of this solvent.

Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs.

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst2 and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst2 in the nM range with best results obtained with peptide 2, that is, IC50 = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC50 = 60 nM.

Beta,beta-disubstituted C- and N-vinylindoles from one-step condensations of aldehydes and indole derivatives.

Direct preparation of beta,beta-disubstituted C- and N-vinylindoles from condensation of aldehydes on indole derivatives is presented. Heating 1-methyl- and 1-benzylindole 3a,b with alkyl and aryl alpha-branched aldehydes and TFA in acetonitrile using microwave irradiation furnished 3-vinylindoles 1a-e in 18-76% yields. Under similar conditions, 3-substituted indoles 4a-c provided N-vinylindoles 2a-h in 16-98% yields.

2-vinylpyrroles and pyrrolo[3,2-d]pyrimidines from direct addition of aldehydes to 4-amino-pyrrole-2-carboxylate derivatives.

A new methodology for the direct preparation of 2-vinylpyrroles is presented. Treatment of 4-amino-pyrrole-2-carboxylates 5a-c and 6a-d with aliphatic aldehydes and TFA furnished 2-vinylpyrroles 2a-k in 9-87% yields. Under similar conditions ureidopyrroles 5a-c reacted with aryl aldehydes to provide pyrrolo[3,2-d]pyrimidines 1a-d in 28-63% yields.

Reversible pegylation prolongs the hypotensive effect of atrial natriuretic peptide.

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.

Deazapurine solid-phase synthesis: combinatorial synthesis of a library of N3,N5,C6-trisubstituted pyrrolo[3,2-d]pyrimidine derivatives on cross-linked polystyrene bearing a cysteamine linker.

Solid-phase methodology for the preparation of pyrrolo[3,2-d]pyrimidine-6-carboxylates with diversity at the N3 pyrmidine nitrogen has now been elaborated to allow for the generation of pyrrolopyrimidine libraries with members possessing diversity at the N3, N5, and C6 positions. The diversification of the N5 position was achieved by treating the parent resin-bound pyrrolo[3,2-d]pyrimidines 3 with an alkyl halide in the presence of Cs2CO3 in DMF. Modification of the C6 carboxylate of resin-bound pyrrolopyrimidines 3-5 was first achieved by hydrolysis of the benzyl ester using LiOH in a mixture of THF/H2O/MeOH. Further alteration of the C6 position of resin-bound pyrrolo[3,2-d]pyrimidine-6-carboxylic acids 6-8 was then performed by activation with triphosgene and treatment with an amine to furnish resin-bound pyrrolo[3,2-d]pyrimidine-6-amides. Twenty-two pyrrolo[3,2-d]pyrimidines 1a-v with different substituents at the N3, N5, and C6 positions were obtained in yields of 21-83% and purities of 61-98% after cleavage from the solid support.

Functional analysis of backbone cyclic peptides bearing the arm domain of the HIV-1 Rev protein: characterization of the karyophilic properties and inhibition of Rev-induced gene expression.

This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin beta in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60-70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed.