Crystalline‐Sponge‐Based Structural Analysis of Crude Natural Product Extracts

Characterization of complex natural product mixtures to the absolute structural level of their components often requires significant amounts of starting materials and lengthy purification process, followed by arduous structure elucidation efforts. The crystalline sponge (CS) method has demonstrated utility in the absolute structure elucidation of isolated organic compounds at miniscule quantities compared to conventional methods. In this work, we developed a new CS‐based workflow that greatly expedites the in‐depth structural analysis of crude natural product extracts. Using a crude extract of the red alga Laurencia pacifica, we showed that CS affinity screening prior to compound isolation enables prioritization of analytes present in the extract, and we subsequently resolved the molecular structures of six sesquiterpenes with stereochemical clarity from around 10 mg crude extract. This study demonstrates a new chemotyping workflow that can greatly accelerate natural product discovery from complex samples.     

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme B12 Catalysis

The crystal structures of the B₁₂‐dependent isomerases (eliminating) diol dehydratase and ethanolamine ammonia‐lyase complexed with adenosylcobalamin were solved with and without substrates. The structures revealed that the peripheral a‐acetamide side chain of the corrin ring directly interacts with the adenosyl group to maintain the group in the catalytic position, and that this side chain swings between the original and catalytic positions in a synchronized manner with the radical shuttling between the coenzyme and substrate/product. Mutations involving key residues that cooperatively participate in the positioning of the adenosyl group, directly or indirectly through the interaction with the a‐side chain, decreased the turnover rate and increased the relative rate of irreversible inactivation caused by undesirable side reactions. These findings guide the engineering of enzymes for improved catalysis and producing useful chemicals by utilizing the high reactivity of radical species.     

Supramolecular assemblies formed by new L-lysine derivatives of viologens

L-Lysine derivatives of viologens form supramolecular assemblies of fibers and ribbons in some aromatic solvents, and the charge separation reaction in these self-assembling systems proceeds with a similar efficiency to the MV2+ system.     

Photocrosslinking reaction of vinyl‐functional polyphenylsilsesquioxane sensitized with aromatic bisazide compounds

Photochemical reactions of aromatic azide groups were applied for a novel photosensitive silicone ladder polymer, that is, partially vinyl‐substituted polyphenylsilsesquioxane sensitized with aromatic bisazide compounds as a photocrosslinker. The photocrosslinking reaction in this system was investigated from the viewpoint of the efficiency of the photocrosslinker, that is, the ratio of the photocrosslinker consumed for crosslinking. The numbers of photodecomposed azide groups and crosslinks in the polymer were determined by Fourier transform infrared measurements. At a higher bisazide concentration, the predominant reaction of nitrenes formed as the intermediary radical by the photolysis of azide was a coupling reaction that could not contribute to the gelation of the polymer. The ratio of the bisazide compound consumed for crosslinking showed the highest value at its concentration of 3 wt % and decreased with the addition of a larger amount. The semiempirical molecular orbital calculations were applied to the theoretical analysis of the photoreaction of nitrenes using phenylnitrene as a model structure. The calculation results indicated that the coupling reaction of nitrenes should proceed more easily than the photocrosslinking reaction in N₂ atmosphere, and the fact that the oxidation of nitrenes should proceed exclusively in the atmosphere including O₂ agreed with the experimental results. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 4196–4205, 2001     

Effects of added metal ions on the interaction between polyvinylpyrrolidone and azo dyes carrying hydroxyl groups

The extent of binding of chrome violet, which is a monoazo dye and involves two hydroxyl groups in o and o′ positions to azo group, by polyvinylpyrrolidone is markedly enhanced in the presence of Co²⁺ ion. The amount of binding in the presence of 1 × 10^?4 mol/L of Co²⁺ ion increases by a factor of about 10 compared to that in the absence of the metal ion. Ni²⁺ and Zn²⁺ ions do not perceptively influence the binding affinity of the dye. Cu²⁺ ion, in contrast, suppresses the binding. To investigate further the action of added metal ions, a cobalt–complex dye was prepared and its binding property for the polymer was compared to that of chrome violet in the presence of metal ions. Some possible mechanisms for the enhancement of chrome violet binding by the addition of Co²⁺ ion are described.     

Photoreaction Cycle of Phoborhodopsin (Sensory Rhodopsin II) from Halobacterium salinarum Expressed in Escherichia coli

Phoborhodopsin (pR; also called sensory rhodopsin II, SRII) is a photoreceptor of negative phototaxis of halobacteria. The studies of photochemical properties of this pigment are not many because the amount of the pigment is small and the stability is low. Recently an expression system of phoborhodopsin from Halobacterium salinarum (called salinarum phoborhodopsin, spR; also HsSRII) in Escherichia coli and purification method has been developed (Mironova et al. [2005] FEBS Lett., 579, 3147–3151), which enables detailed studies on the photochemical properties of spR. In the present work, the photoreaction cycle of E. coli-expressed spR was studied by low-temperature spectroscopy and flash photolysis. Formations of K-, M-, O-like intermediates and P480 were reconfirmed as reported previously. New findings are as follows. (1) The K-like intermediate (P500) was a mixture of two photoproducts. (2) Formation of L-like intermediate (P482) was observed by low-temperature spectroscopy and flash photolysis at room temperature. (3) On long irradiation of spR at 20°C, formation of a new photoproduct P370 was observed and it decayed to the original spR in the dark with a decay half time of 190 min. Based on these results the similarities and dissimilarities between spR and ppR are discussed.     

Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.