Synthesis of trans-Tetrafluoro(trifluoromethyl)-λ6-sulfanyl (CF3SF4)-Containing Olefins via Cross Metathesis.

The cross-metathesis reactions of trans-tetrafluoro(trifluoromethyl)-λ6-sulfanyl (CF3SF4)-containing olefins expand the repertoire of synthetic transformations of CF3SF4-substituted molecules. Treatment of a primary alkene and 3-CF3SF4-propene with a second-generation Hoveyda-Grubbs catalyst yielded the cross-metathesis product in good yield under very mild conditions (room temperature). CF3SF4-propene undergoes cross metathesis with substrates containing electron-withdrawing groups or electron-donating groups at room temperature or under dichloromethane reflux. The formation of the CF3SF4-propene homodimer and the utility of that dimer to undergo selective cross-metathesis reactions are described.

N-(2-Tetrafluoro(trifluoromethyl)-λ6-sulfanyl(CF3SF4)-ethyl) Amines: The Influence of the CF3SF4 Group on Lipophilicity and pKa.

A practical synthetic path for the preparation of trans-CF3SF4-substituted amines has been described. Primary and secondary amines bearing a variety of different functional groups including amino acids, cyclic amines, and nucleosides were prepared. The desired amines were synthesized under mild conditions. The influence of the CF3SF4-group on the pKa and log D of a standard amine was established. The unusual conformation of the trans-CF3SF4-substituted tosylate has been verified via its crystal structure.

Resonance Energy Transfer in a Genetically Engineered Polypeptide Results in Unanticipated Fluorescence Intensity.

The fluorescence intensity of a C-terminal acceptor chromophore, N-(7-dimethylamino-4-methyl coumarin (DACM), increased proportionally with 280 nm irradiation of an increasing number of donor tryptophan residues located on a ß-sheet forming polypeptide. The fluorescence intensity of the acceptor chromophore increased even as the length of the ß-sheet edge approached 256 Å, well beyond the Förster radius for the tryptophan-acceptor chromophore pair. The folding of the peptides under investigation was verified by circular dichroism (CD) and deep UV resonance Raman experiments. Control experiments showed that the enhancement of DACM fluorescence occurred concomitantly with peptide folding. In other control experiments, the DACM fluorescence intensity of the solutions of tryptophan and DACM did not show any enhancement of DACM fluorescence with increasing tryptophan concentrations. Formation of fibrillar aggregates of the substrate peptides prepared for the fluorescence studies was undetectable by thioflavin T (ThT) fluorescence.

The secondary structure of a heptapeptide containing trifluoromethyl-λ6-tetrafluorosulfanyl substituted amino acids.

Site specific introduction of the polar hydrophobic trifluoromethyl-λ6-tetrafluorosulfanyl (CF3SF4) group can effectively control the secondary structure of a heptapeptide, the minimum repeat unit of an α-helix. The structural influence of CF3SF4-containing amino acid on the heptapeptide was established using NMR methods.

The control of stereochemistry by the pentafluorosulfanyl group.

The influence of pentafluorosulfanylation on biological activity has been revealed in numerous comparative studies of biologically active compounds, but considerably less is known about the influence of pentafluorosulfanylation on reactivity. Among the distinctive properties of the pentafluorosulfanyl group is the profound dipole moment that results from introduction of this substituent. It has been shown that dipolar effects coupled with the steric demand of the SF5 group may be employed to influence the stereochemistry of reactions, especially those processes with significant charge separation in the transition state. The Staudinger ketene-imine cycloaddition reaction is an ideal platform for investigation of dipolar control of diastereoselectivity by the pentafluorosulfanyl group.

Studies of the Binding of Modest Modulators of the Human Enzyme, Sirtuin†6, by STD NMR.

Pyrazinamide (PZA), an essential constituent of short-course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite the structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 by different mechanisms and through different binding sites, as suggested by saturation transfer difference (STD) NMR. Available experimental evidence, such as that derived from crystal structures and kinetic experiments, has been of only limited utility in elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For instance, crystallographic structural analysis of sirtuin binding sites does not help us understand important differences in binding affinities among sirtuins or capture details of such dynamic process. Hence, STD NMR was utilized throughout this study. Our results not only agreed with the binding kinetics experiments but also gave a qualitative insight into the binding process. The data presented herein suggested some details about the geometry of the binding epitopes of the ligands in solution with the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates might be possible. These derivatives might be accessible by employing the malleability of this scaffold to assist in the identification by STD NMR of the motifs that interact with the apo- and holoenzymes in solution.

The role of proline-containing peptide triads in ß-sheet formation: A kinetic study.

The design of biomimetic materials through molecular self-assembly is a growing area of modern nanotechnology. With problems of protein folding, self-assembly, and sequence-structure relationships as essential in nanotechnology as in biology, the effect of the nucleation of ß-hairpin formation by proline on the folding process has been investigated in model studies. Previously such studies were limited to investigations of the influence of proline on the formation of turns in short peptide sequences. The effect of proline-based triads on the folding of an 11-kDa amyloidogenic peptide GH6[(GA)3GY(GA)3GE]8 GAH6 (YE8) was investigated by selective substitution of the proline-substituted triads at the γ-turn sites. The folding and fibrillation of the singly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]7(GA)3GY(GA)3PD-GAH6 (8PD), and doubly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD-GAH6 (4,8PD), were directly monitored by circular dichroism and deep UV resonance Raman and fluorescence spectroscopies. These findings were used to identify the essential folding domains, i.e., the minimum number of ß-strands necessary for stable folding. These experimental findings may be especially useful in the design and construction of peptidic materials for a wide range of applications as well as in understanding the mechanisms of folding critical to fibril formation.

Chimera-induced folding: implications for amyloidosis.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

Preparation and characterization of alkenyl aryl tetrafluoro-λ(6) -sulfanes.

Substituted alkenyl aryl tetrafluoro-λ(6) -sulfanes have been prepared by the direct addition of readily accessible chlorotetrafluorosulfanyl arenes to primary alkynes. Substitution of an apical fluorine of the pentafluorosulfanyl group enables modulation of the reactivity of this little explored functional group while at the same time facilitating the direct investigation of aryl substituent effects on the aryl tetrafluorosulfanyl-substituted products.

Diastereoselectivity in the Staudinger reaction of pentafluorosulfanylaldimines and ketimines.

ß-Lactams were diastereoselectively formed by the reaction of SF5-containing aldimines, or an SF5-containing ketimine, with benzyloxyketene in a conrotatory ring closure process. Imine formation and cyclization were possible in spite of the acidification of protons on the carbon bound to SF5. The reactions of the aldimines demonstrated very good 1,2-lk diastereoselectivity, however lack of stereochemical control of the C-N ketimine geometry was reflected in the stereochemistry of the product ß-lactam. Cyclization of imines with a stereogenic center bearing SF5 was reflected in the 1,2-lk,lk selectivity of the ß-lactam.

Aggregation in Aqueous Solutions of 2-(Tetrafluoro(trifluoromethyl)-λ6-sulfanyl-ethan-1-ol (CF3SF4-ethanol)): A Comparison with Aqueous Trifluoroethanol and Hexafluoroisopropanol Using Molecular Dynamics Simulations and Dynamic Light Scattering Experiments.

2-Tetrafluoro(trifluoromethyl)-λ6-sulfanylethan-1-ol (CF3SF4-ethanol) combines the polar hydrophobicity of tetrafluoro(trifluoromethyl)-λ6-sulfanyl (CF3SF4) group with the polarity of simple alcohols. The properties of aqueous solutions of the well-known fluorinated alcohols 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) were compared with those of aqueous solutions of the novel CF3SF4-ethanol. Those properties were computed using all atom molecular dynamics simulations with OPLS-compatible parameters. DFT ab initio calculations were used to accurately describe the nonsymmetrical, hypervalent sulfur in CF3SF4-ethanol. Although the molecular and conformational characteristics of CF3SF4-ethanol are like those of both TFE and HFIP, the greater hydrophobicity and lower polarity of CF3SF4-ethanol resulted in solution phase aggregation at a much lower concentration. The properties computed for TFE and HFIP in this work were consistent with published computational and experimental studies. CF3SF4-ethanol is predicted to be environmentally benign and hence an excellent green solvent candidate while possessing many of the same properties as TFE or HFIP.

Fibrillation mechanism of a model intrinsically disordered protein revealed by 2D correlation deep UV resonance Raman spectroscopy.

Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of ß-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and ß-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.

Activity of 5-chloro-pyrazinamide in mice infected with Mycobacterium tuberculosis or Mycobacterium bovis.

BACKGROUND & OBJECTIVES: Pyrazinamide is an essential component of first line anti-tuberculosis regimen as well as most of the second line regimens. This drug has a unique sterilizing activity against Mycobacterium tuberculosis. Its unique role in tuberculosis treatment has lead to the search and development of its structural analogues. One such analogue is 5-chloro-pyrazinamide (5-Cl-PZA) that has been tested under in vitro conditions against M. tuberculosis. The present study was designed with an aim to assess the activity of 5-Cl-PZA, alone and in combination with first-line drugs, against murine tuberculosis. METHODS: The minimum inhibitory concentration (MIC) of 5-Cl-PZA in Middlebrook 7H9 broth (neutral pH) and the inhibitory titre of serum from mice that received a 300 mg/kg oral dose of 5-Cl-PZA 30 min before cardiac puncture were determined. To test the tolerability of orally administered 5-Cl-PZA, uninfected mice received doses up to 300 mg/kg for 2 wk. Four weeks after low-dose aerosol infection either with M. tuberculosis or M. bovis, mice were treated 5 days/wk with 5-Cl-PZA, at doses ranging from 37.5 to 150 mg/kg, either alone or in combination with isoniazid and rifampicin. Antimicrobial activity was assessed by colony-forming unit counts in lungs after 4 and 8 wk of treatment. RESULTS: The MIC of 5-Cl-PZA against M. tuberculosis was between 12.5 and 25 µg/ml and the serum inhibitory titre was 1:4. Under the same experimental conditions, the MIC of pyrazinamide was >100 µg/ml and mouse serum had no inhibitory activity after a 300 mg/kg dose; 5-Cl-PZA was well tolerated in uninfected and infected mice up to 300 and 150 mg/kg, respectively. While PZA alone and in combination exhibited its usual antimicrobial activity in mice infected with M. tuberculosis and no activity in mice infected with M. bovis, 5-Cl-PZA exhibited antimicrobial activity neither in mice infected with M. tuberculosis nor in mice infected with M. bovis. INTERPRETATION & CONCLUSION: Our findings showed that 5-Cl-PZA at doses up to 150 mg/kg was not active in chronic murine TB model. Further studies need to be done to understand the mechanism and mode of inactivation in murine model of tuberculosis.

Analogs of the antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty acid synthase I.

Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.

Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I.

Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.

Mutually exclusive genotypes for pyrazinamide and 5-chloropyrazinamide resistance reveal a potential resistance-proofing strategy.

The pyrazinamide (PZA) analog 5-chloropyrazinamide (5-Cl PZA) is active against mycobacterial species, including PZA-resistant strains of Mycobacterium tuberculosis. In M. smegmatis, overexpression of the type 1 fatty acid synthase (FAS I) confers resistance to 5-Cl PZA, a potent FAS I inhibitor. Since M. tuberculosis and M. bovis cannot tolerate FAS I overexpression, 5-Cl PZA resistance mutations have yet to be described for tubercle bacilli. In an attempt to identify other factors that govern the activity of 5-Cl PZA, we selected for 5-Cl PZA-resistant isolates from a library of transposon-mutagenized M. smegmatis isolates. Here, we report that increased expression of the M. smegmatis pyrazinamidase PzaA confers resistance to 5-Cl PZA and susceptibility to PZA in M. smegmatis, M. tuberculosis, and M. bovis. In contrast, while ectopic overexpression of the M. tuberculosis pyrazinamidase PncA increases PZA susceptibility, this amidase does not mediate resistance to 5-Cl PZA. We conclude that PncA-independent turnover of 5-Cl PZA represents a potential mechanism of resistance to this compound for M. tuberculosis, which will likely translate into enhanced PZA susceptibility. Thus, countersusceptibility can be manipulated as a resistance-proofing strategy for PZA-based compounds when these agents are used simultaneously.

Charge distribution and amyloid fibril formation: insights from genetically engineered model systems.

The influence of electrostatic interactions on protein amyloidogenesis has been investigated using de novo designed repetitive polypeptides YEHK21 [GH6[(GA)3GY(GA)3GE(GA)3GY(GA)3GE]21GAH6] and YE8 [GH6[(GA)3GY(GA)3GE]8GAH6]. The beta-sheet forming polypeptides were designed with identical beta-strands but with variable substitution at the turns that enable precise location of charged residues (Topilina et al. Biopolymers 2007, 86 (4), 261-264; Topilina et al. Biopolymers 2010, submitted for publication; Topilina et al. Biomacromolecules 2006, 7 (4), 1104-11). Solubility, folding, and aggregation of YEHK21 and YE8 were shown to be controlled by charge distribution. Under those conditions favoring the development of charge, YEHK21 and YE8 have significant propensities to form intermolecular beta-sheet assemblies illustrating the potential of charged polypeptide chains to form ordered amyloid aggregates even in the absence of additional environmental factors such as the presence of polyelectrolytes, salts, and so on.

The antituberculosis drug pyrazinamide affects the course of cutaneous leishmaniasis in vivo and increases activation of macrophages and dendritic cells.

Antileishmanial therapy is suboptimal due to toxicity, high cost, and development of resistance to available drugs. Pyrazinamide (PZA) is a constituent of short-course tuberculosis chemotherapy. We investigated the effect of PZA on Leishmania major promastigote and amastigote survival. Promastigotes were more sensitive to the drug than amastigotes, with concentrations at which 50% of parasites were inhibited (MIC(50)) of 16.1 and 8.2 microM, respectively (48 h posttreatment). Moreover, 90% of amastigotes were eliminated at 120 h posttreatment, indicating that longer treatments will result in parasite elimination. Most strikingly, PZA treatment of infected C57BL/6 mice resulted in protection against disease and in a 100-fold reduction in the parasite burden. PZA treatment of J774 cells and bone marrow-derived dendritic cells and macrophages increased interleukin 12, tumor necrosis factor alpha, and activation marker expression, as well as nitric oxide production, suggesting that PZA enhances effective immune responses against the parasite. PZA treatment also activates dendritic cells deficient in Toll-like receptor 2 and 4 expression to initiate a proinflammatory response, confirming that the immunostimulatory effect of PZA is directly caused by the drug and is independent of Toll-like receptor stimulation. These results not only are strongly indicative of the promise of PZA as an alternative antileishmanial chemotherapy but also suggest that PZA causes collateral immunostimulation, a phenomenon that has never been reported for this drug.

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils.

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.