Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.

Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound 8e displayed an antivirally effective concentration of 0.4 microM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index > or =50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound 8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.

Symmetric paramagnetic diene 6 was prepared and proved to be a useful intermediate in 1,4-addition, cheletropic, and Diels-Alder reactions. These methods were used for the synthesis of both aromatic 10, 12 heteroaromatic 15, 23 and heterocyclic ring fused nitroxide rings 7, 14, 16, 25a, 25b. The symmetric dibromo compound 20 led to homobifunctional spin label reagents 21, 22b, 24a, 24b, 27a, 27b heterobifunctional reagents 26a, 28a and to double sensor reagents 26b, 28b.The Diels-Alder reaction is an important tool in organic chemistry for construction of six-membered rings. A great variety of carbo-and heterocyclic ring systems have been synthesized in this way. 1 The synthesis of paramagnetic asymmetric six-membered 2 and five-membered 3 (17) dienes is well documented and their Diels-Alder reactions were also reported. The challenge was the synthesis of paramagnetic symmetric pyrrolidine diene 6 which was expected to be a versatile synthetic intermediate. It is interesting to note that the synthesis of the sterically hindered amine precursor of target molecule 6 was reported 30 years ago, 4 but as far as we know the amine was not oxidized to nitroxide. Stable paramagnetic nitroxides are widely used as spin labels, 5 MRI reagents 6 or co-oxidants 7 and their pharmacological properties are also of interest. 8In this paper we report on the synthesis and reactions of a paramagnetic symmetric pyrrolidine diene 6 in the presence of a paramagnetic free radical centre. Oxidation of γ-nitro ester 1, which was synthesized by Michael addition of nitromethane to an α,β-unsaturated ester, 9 with Nmethylmorpholin-N-oxide (NMO) in the presence of tetrapropylammonium perruthenate (TPAP) resulted not only in the conversion of the nitro group to a formyl group 10 but also in the oxidation of the pyrrolidine ring to pyrroline by forming the double bond yielding the α,β-unsaturated aldehyde ester 2. We found a simpler and cheaper oxidation method with KMnO 4 in basic medium 11 which gave saturated aldehyde ester 3 and also biradical 4 as a byproduct. This can be attributed to the in situ Henry nitro condensation 12 between 1 and 3 followed by oxidation. Treatment of compound 3 with NMO/TPAP yielded 2, demonstrating that a Ley-Griffith oxidation reagent 13 was capable of introducing the double bond. Compound 3 was easily reduced with NaBH 4 in EtOH to saturated paramagnetic 1,4-diol 5a which could be converted to the diiodide 5b through mesylate. The diiodide 5b was converted to the thiol specific reversible bisthiosulfonate reagent 5c by cautious heating of compound 5b with NaSSO 2 Me in DMSO. Elimination of HI from the diiodide 5b was carried out in aqueous NaOH/EtOH solution with heating resulting in the desired diene 6 (Scheme 1).Reagents and conditions: a) NMO (1.5 equiv)/AgOAc (2 equiv)/ K 2 CO 3 (5 equiv)/TPAP (0.1 equiv)/MeCN, 40°C, 12 h (62-75%); b) KOH (1.2 equiv)/MeOH, 0°C, 30 min, then KMnO 4 (0.7 equiv)/ MgSO 4 (1 equiv)/H 2 O, 0°C, 2 h (3: 37%, 4: 59%); c) NaBH 4 (3 equiv)/EtOH, 30 min, r.t. (80%); d) i: MsCl (2.2 equ...