Spectroscopic studies of the corrinoid/iron-sulfur protein from Moorella thermoacetica

Methyl transfer reactions are important in a number of biochemical pathways. An important class of methyltransferases uses the cobalt cofactor cobalamin, which receives a methyl group from an appropriate methyl donor protein to form an intermediate organometallic methyl-Co bond that subsequently is cleaved by a methyl acceptor. Control of the axial ligation state of cobalamin influences both the mode (i.e., homolytic vs heterolytic) and the rate of Co-C bond cleavage. Here we have studied the axial ligation of a corrinoid iron-sulfur protein (CFeSP) that plays a key role in energy generation and cell carbon synthesis by anaerobic microbes, such as methanogenic archaea and acetogenic bacteria. This protein accepts a methyl group from methyltetrahydrofolate forming Me-Co(3+)CFeSP that then donates a methyl cation (Me) from Me-Co(3+)CFeSP to a nickel site on acetyl-CoA synthase. To unambiguously establish the binding scheme of the corrinoid cofactor in the CFeSP, we have combined resonance Raman, magnetic circular dichroism, and EPR spectroscopic methods with computational chemistry. Our results clearly demonstrate that the Me-Co3+ and Co2+ states of the CFeSP have an axial water ligand like the free MeCbi+ and Co(2+)Cbi+ cofactors; however, the Co-OH2 bond length is lengthened by about 0.2 angstroms for the protein-bound cofactor. Elongation of the Co-OH2 bond of the CFeSP-bound cofactor is proposed to make the cobalt center more "Co1+-like", a requirement to facilitate heterolytic Co-C bond cleavage.     

Radiochemical approaches to fullerene chemistry

Nuclear and radiochemistry offers special and attractive possibilities for exploring the fascinating world of closed-shell carbon cages called fullerenes. This review presents a panoramic view on the use of radioactivity, nuclear irradiation and instrumental nuclear techniques in the elucidation of the structure and composition of fullerenes and their compounds.     

The vaccinia virus HindIII K fragment encodes a novel protein belonging to the serpin superfamily

The 1983-base pair nucleotide sequence of the EcoRI-HindIII fragment of vaccinia virus Tiantan strain HindIII K clone is determined by the dideoxy chain termination method. A search in the NBRF protein sequence database using FASTA and other microcomputer programs reveals that several proteins belonging to the serpin (serine protease inhibitor) superfamily have striking similarities to the protein encoded by the HindIII K1 ORF. On the basis of the dot-matrix analysis and sequence alignment, the K1-encoded protein is shown as a novel member of the serpin superfamily. The putative reactive site and switch sequence of this novel serpin are then compared with those of other serpins. The probable evolutionary and possible functional relationships are discussed.     

Quantum chemical approaches to the explanation of differences in NO-donor activity of iron-sulfur nitrosyl complexes

The geometries and electronic structures of iron-sulfur nitrosyl complexes with azaheterocyclic thiols with μ-SCN bridging ligands, [Fe₂(SCN₄Ph)₂(NO)₄] (1) and [Fe₂(SCN₄Me)₂(NO)₄] (2), were calculated within the framework of the density functional theory (DFT) with the BP86, TPSS, B3LYP, and OPBE functionals in order to explain differences in their NO-donor activities. Chemical bonding in the complexes was analyzed using the NBO approach, according to Mulliken, and using the Voronoi scheme. NBO analysis of complex 1 revealed a strong interaction between orbitals of the lone electron pairs of the Fe atom and the antibonding orbitals of the Fe-N bond which leads to strengthening of the Fe-NO bond. In complex 2, the Fe-NO bond is more polar than in complex 1.     

Spectroscopic diagnostics of organic chemistry in the protostellar environment

A combination of astronomical observations, laboratory studies, and theoretical modelling is necessary to determine the organic chemistry of dense molecular clouds. We present spectroscopic evidence for the composition and evolution of organic molecules in protostellar environments. The principal reaction pathways to complex molecule formation by catalysis on dust grains and by reactions in the interstellar gas are described. Protostellar cores, where warming of dust has induced evaporation of icy grain mantles, are excellent sites in which to study the interaction between gas phase and grain-surface chemistries. We investigate the link between organics that are observed as direct products of grain surface reactions and those which are formed by secondary gas phase reactions of evaporated surface products. Theory predicts observable correlations between specific interstellar molecules, and also which new organics are viable for detection. We discuss recent infrared observations obtained with the Infrared Space Observatory, laboratory studies of organic molecules, theories of molecule formation, and summarise recent radioastronomical searches for various complex molecules such as ethers, azaheterocyclic compounds, and amino acids.     

Spectroscopic methods in bioinorganic chemistry: blue to green to red copper sites

A wide variety of spectroscopic methods are now available that provide complimentary insights into the electronic structures of transition-metal complexes. Combined with calculations, these define key bonding interactions, enable the evaluation of reaction coordinates, and determine the origins of unique spectroscopic features/electronic structures that can activate metal centers for catalysis. This presentation will summarize the contributions of a range of spectroscopic methods combined with calculations in elucidating the electronic structure of an active site using the blue copper site as an example. The contribution of electronic structure to electron-transfer reactivity will be considered in terms of anisotropic covalency, electron-transfer pathways, reorganization energy, and protein contributions to the geometric and electronic structures of blue-copper-related active sites.     

New analytical approaches in clinical chemistry

Millions of chemical analyses are performed in clinical laboratories every day, and the demand of the medical profession for increasingly accurate and specific assays continues to grow. This has resulted in the development of many new techniques and, just as important, the use of proven analytical systems in novel ways.     

Spectroscopic approaches to the study of the interaction of aluminum with humic substances

Aluminum ion (Al³⁺) in the ‘free’ (aquo) state is becoming increasingly prevalent in environmental waters, especially fresh waters, as a consequence of acid rain and other environmental processes. As Al³⁺ ion is known to affect markedly a wide range of biological systems, and since the presence of Al³⁺ in humans has been linked to a number of human diseases, it is important to understand the speciation of Al³⁺ ion in natural waters. Since some of the most important complexation agents for Al³⁺ in both fresh and sea waters are members of the complex humic substances group, it is important to understand the manner in which Al³⁺ interacts with this class of molecules, especially since binding of Al³⁺ to these molecules can effectively increase the bioavailability of this toxic metal ion to biological systems. The objective of this review is to present the current state of our understanding of aqueous aluminum complexation with the most acidic members (and therefore the most likely candidates for serving as Al³⁺ complexing agents) of the humic substances group, the fulvic acids. Much of the current knowledge has been revealed by comprehensive fluorescence titration analyses. Some additional information has come from other experimental approaches, including infrared spectroscopy, nuclear magnetic resonance spectroscopy, and a variety of electrochemical approaches. In this review, we also report on the results of our recent fluorescence and IR spectroscopy survey of the interaction of metals from of all three Nieboer and Richardson categories of environmental metals (Class A, Class B and Intermediate Class) with the fulvic acid sub-group of the humic substances. This has proven helpful in understanding some of the unique spectral behaviors of the Al³⁺-fulvic acid complex vis-a-vis fulvic acid complexes with many other metal ions. The results of our fluorescence and IR experiments with the model compounds, such as salicylic and phthalic acids, have allowed confirmation of the important roles played by both salicylic acid-like sites and phthalic acid-like sites in the unique complexation of Al³⁺ to humic substances, and help to explain some of the observed spectroscopic changes associated with Al³⁺ ion complexation to humic material. From the current work, it seems clear that major sources of the deviation in spectral properties between Al³⁺ and many other metal ions (across all three Nieboer and Richardson categories) are the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (i.e. a very high ionic index combined with a relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

New synthetic approaches to development of the chemistry of pentaerythritol bicyclophosphite

Syntheses of the original compact molecular systems were developed and the ways of their practical use were examined. A group of new substances was synthesized on the basis of available penta-erythritol bicyclophosphite and thionophosphate, including respective ammonium salts, phosphocholines, a complex compound of molybdenum, and a hydrophosphoryl compound.     

Genetic approaches to protein purification

A gene fusion system based on the protein A gene from Staphylococcus aureus has been developed to facilitate purification of recombinant proteins, both in large and small scale. Due to the strong interaction with IgG, it is possible to recover gene products fused to various protein A derivatives, in a one-step procedure with high yield and in purity. Site-directed mutagenesis was used to introduce enzymatic and chemical cleavage sites at the fusion point between the protein A derivative and the desired protein. The protein A “tail” can thereby be removed from the affinity purified fusion protein by the appropriate cleavage, releasing biologically active molecules. Recently, the system was improved by designing a synthetic DNA fragment encoding two IgG-binding domains derived from staphylococcal protein A which are resistant to various chemical cleavages. The gene fusion product is secreted to the culture medium of E.coli and can be recovered simply by passing the clarified culture medium through an IgG Fast Flow Sepharose. The system has been used to immobilize enzymes, to obtain monoclonal and polyclonal antibodies and to produce biologically active human peptide hormones in pilot plant scale.     

"Click chemistry"en route to pseudo-starch

Rapid assembly of starch fragment analogues was achieved using "click chemistry". Specifically, two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to a maltose core were synthesized using Cu(i)-catalyzed [3 + 2] dipolar cycloaddition of azido saccharides and 6,6'- and 4',6'-dipropargylated p-methoxyphenyl maltoside.     

Exploiting PdII and TiIII chemistry to obtain gamma-dioxygenated terpenoids: synthesis of rostratone and novel approaches to aphidicolin and pyripyropene A

In nature there are several terpenoids with a characteristic gamma-dioxygenated system on the A ring, and many of them show interesting pharmacological properties. We have developed a novel strategy for the synthesis of these terpenoids involving three stages: (a) the selective epoxidation of commercial polyenes, (b) titanium(III)-catalyzed cyclization of the epoxypolyprenes thus obtained, and (c) Pd-mediated remote functionalization of the equatorial methyl group attached at C-4 on ring A of the cyclic terpenoid thus formed. This strategy has proved to be useful for the synthesis of the natural labdane rostratone (1) and related terpenoids, as well as for advanced synthetic approaches toward the pharmacologically active products aphidicolin (2) and pyripyropene A (3).     

Green chemistry approaches for the purification of pharmaceuticals

To enable R&D of new drug products with a more green process emphasis, a full understanding of the impact of chromatography in the discovery phase of drug development is crucial. With large quantities of small scale (<1000 mg) separations typically evaluated and purified, a chromatographic technique needs to be easily integrated into workflows and readily robust. At Novartis, integration of supercritical fluid chromatography (SFC) has allowed for increases in productivity, decreases in toxic/hazardous solvent consumption and a more green approach to chromatography. The purpose of this review is to introduce the reader to new techniques available to organizations for the analysis and purification of mg-kg scale purifications that are routinely handled within discovery. Evaluation of a variety of techniques available to the industry has led to the implementation of SFC, with some of the pros of each technique noted below: An overview of enantiomeric purification methodologies is highlighted below in Table 1.     

New "green" approaches to the synthesis of pyrazole derivatives

A novel approach to the synthesis of pyrazole derivatives from tosylhydrazones of alpha,beta-unsaturated carbonyl compounds possessing a beta-hydrogen is proposed, exploiting microwave (MW) activation coupled with solvent free reaction conditions. The cycloaddition was studied on three ketones (trans-4-phenyl-3-buten-2-one, beta-ionone and trans-chalcone). The corresponding 3,5-disubstituted-1H-pyrazoles were obtained in high yields and after short reaction times. In order to simplify and point out the green chemistry features of the method, a further improvement was achieved under the same catalytic conditions with a "one pot" synthesis of these heterocyclic compounds, starting directly from their carbonyl precursors via tosylhydrazones generated in situ. For an exhaustive study, the dielectric properties of the solid reaction mixtures were also measured, in order to obtain input data for the numerical simulation of their heating behaviour in the single mode MW cavity which was used for experimental work. In order to supply a valid methodology and tool for measuring the environmental impact, a comparative study between the synthetic route proposed and the classical synthetic route has been carried out.     

Orthogonal approaches to the simultaneous and cascade functionalization of macromolecules using click chemistry

The development of selective chemistries that are orthogonal to the diverse array of functional groups present in many polymeric systems is becoming an important tool for the synthesis and use of macromolecules in fields ranging from biomedical devices to nanotechnology. By combining copper-catalyzed cycloaddition chemistry with other synthetic transformations such as esterification, amidation, etc., highly efficient and modular simultaneous and cascade functionalization strategies have been developed. These single-step strategies for preparing multifunctional macromolecules represent a significant advance as compared to traditional multistep approaches, and the utility of these concepts is demonstrated by selective preparation of a diverse range of orthogonally functionalized vinyl polymers.     

Diazo chemistry in the access to novel fatty acids linked to spiro-fused oxetane-pyrazolone scaffold

A novel free fatty acid mimetic based on the spirocyclic oxetane pyrazolone 1-oxa-6,7-diazaspiro[3.4]oct-7-en-5-one scaffold has been obtained in seven steps. The key stages are based upon the toolbox of diazo chemistry, including the F SAFE diazo transfer and Rh^II-catalyzed O–H insertion followed by base-triggered oxetane closing.