This work describes the chemistry and methodology for constructing multilayers of bis-biotinylated dsDNA on metal substrates after enzyme cleavage and demonstrates its use for amplified microgravimetric and impedimetric analyses of anticancer drug, cisplatin. Specific chemical modification of dsDNA prior to immobilization was achieved via a bisulfite-catalyzed transamination of cytosine after endonuclease cleavage of plasmid DNA. The specificity of the reaction of cytosine residues at ss- versus dsDNA loci after endonuclease cleavage was characterized using circular dichroism, mass spectrometry, and absorption spectrophotometry. The biotinylated dsDNA consisting of 2961 base pairs was then used as a ligand at avidin-modified gold electrodes. Ac impedance spectroscopy and quartz crystal microbalance measurements clearly showed that the response to cisplatin increased linearly with target concentrations. The impedance spectroscopy resulted in a detection limit of 1 nM and a surface density of 4.8 × 10¹³ molecules/0.1 cm². The immobilization of dsDNA on surfaces is a significant improvement over existing approaches in that it enables the attachment of long pieces of unmodified double-stranded DNA via a simple biotinylation step. The immobilization technique provides a generic approach for dsDNA-based sensor development and for monitoring DNA−analyte interactions.

¹⁵N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F_exch) and unfolded (U_exch) states under nondenaturing conditions in a ratio of 2:1 at 20 °C, with an average exchange rate constant, k_ex, of 2.2 s^-1 (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U_exch ensemble there is a contiguous stretch of residues undergoing conformational exchange on a μs/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F_exch ↔ U_exch conformational exchange process and the μs/ms exchange event within the U_exch state contribute to the ¹⁵N line width and can be analyzed using CPMG-based ¹⁵N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental ¹⁵N relaxation dispersion data with results from an ¹⁵N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727−734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.

Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply ¹³C-labeled samples of d-glucose. The ¹³C incorporation pattern within pyridoxamine dihydrochloride, established by ¹³C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli. Thus, the origin of the carbon skeleton of vitamin B₆ in yeast differs substantially from its origin in E. coli. In particular, in yeast the distribution of ¹³C within the C₅ chain C-2‘,2,3,4,4‘ of pyridoxamine corresponds to the distribution of ¹³C within the C₅ chain C-1,2,3,4,5 of the ribose component of cytidine. It follows that the C₅ chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate. By contrast, in E. coli the C₅ chain of pyridoxine is derived from 1-deoxy-d-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.

The distribution of the final base damage was determined within isolated DNA exposed to pulses of 266 nm laser light. Studied lesions included oxidation products arising from biphotonic ionization of DNA bases and pyrimidine dimeric photoproducts arising from monophotonic processes. The distribution of the latter class of damage was found to be correlated with the stability of the DNA duplex. The quantum yield for formation of 8-oxo-7,8-dihydroguanine was much higher than that of other oxidized nucleosides arising from the degradation of thymine and adenine. This observation, together with the shape of the intensity dependence curves, provided evidence for the occurrence of charge-transfer processes within DNA. In addition, increase in the ionic strength of the irradiated DNA and stabilization of the DNA duplex were found to induce a drastic decrease in the yield of thymine and adenine oxidation products. Concurrently, an increase in the yield of 8-oxo-7,8-dihydroguanine was observed. This was rationalized in terms of an increase in the overall charge-transfer efficiency. Therefore, it may be concluded that stabilization of the double-helix favors charge-transfer process toward guanine bases.

Cell-permeable small molecules are powerful tools for unraveling complex cellular pathways. We demonstrate that nuclear hormone receptors can be engineered through mutagenesis to create orthogonal ligand−receptor pairs to control transcription. Mutated residues in the retinoid X receptor (RXR) were chosen from structural analysis of RXR and the retinoic acid receptor (RAR) ligand binding domains. The potential ligands screened for activation of variant receptors are “near drugs”compounds synthesized during structure−activity studies that are structurally similar to an approved drug yet inactive on the wild-type receptor. One variant, Q275C;I310M;F313I, is poorly activated by ligands for the wild-type receptor but is activated by a “near drug”, fulfilling the criteria of an orthogonal ligand−receptor pair. These experiments demonstrate that nuclear hormone receptors are well suited to supply orthogonal ligand−receptor pairs for experimental biology, biotechnology, and gene therapy. Our findings also demonstrate the general principle that inactive compounds synthesized during drug discovery can be combined with mutant proteins to rapidly create new tools for controlling cellular processes.

The boron-containing π-conjugated systems, including tri(9-anthryl)borane (1) and tris[(10-dimesitylboryl)-9-anthryl]borane (2), have been investigated as a new type of fluoride chemosensor. Upon complexation of 1 with a fluoride ion, a significant color change from orange to colorless was observed and, in the UV−visible absorption spectra, the characteristic band of 1 at 470 nm disappeared and new bands around 360−400 nm assignable to π−π* transitions of the anthryl moieties were observed. This change can be rationalized as a result of the interruption of the π-conjugation extended through the vacant p-orbital of the boron atom by the formation of the corresponding fluoroborate. The binding constant of compound 1 with the fluoride ion was quite high [(2.8 ± 0.3) × 10⁵ M^-1], whereas 1 only showed small binding constants with AcO^- and OH^- of around 10³ M^-1 and no sensitivity to other halide ions such as Cl^-, Br^-, and I^-, thus demonstrating its selective sensing ability to the fluoride ion. In contrast to the monoboron system 1, compound 2 having four boron atoms showed multistage changes in the absorption spectra by the stepwise complexation with fluoride ions.

A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinones, all shows trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control found in actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tü22 was studied by functional expression of the potentially relevant ketoreductase genes, actIII, actVI-ORF1, gra-ORF5, and gra-ORF6. A common bicyclic intermediate was postulated to undergo stereospecific reduction to provide either the 3-(S) or the 3-(R) configuration of an advanced intermediate, 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid (DNPA). Combinations of the four ketoreductase genes were coexpressed with the early biosynthetic genes encoding a type II minimal polyketide synthase, aromatase, and cyclase. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. Out of the various recombinants carrying the relevant ketoreductases, the set of gra-ORF5 and -ORF6 under translational coupling (on pIK191) led to the most efficient production of (R)-DNPA as a single product, implying a possible unique cooperative function whereby gra-ORF6 might encode a “guiding” protein to control the regio- and stereochemical course of reduction at C-3 catalyzed by the gra-ORF5 protein. Updated BLAST-based database analysis suggested that the gra-ORF6 product, a putative short-chain dehydrogenase, has virtually no sequence homology with the actVI-ORF1 protein, which was previously shown to determine the 3-(S) configuration of DNPA in ACT biosynthesis. This demonstrates an example of opposite stereochemical control in antibiotic biosynthesis, providing a key branch point to afford diverse chiral metabolic pools.

The stereospecific total synthesis of (±)-thielocin A1β has been achieved from the common intermediate ethyl 5-formyl-2,4-dihydroxy-3,6-dimethyl benzoate (8). The racemic synthesis was achieved based on the key reaction of a 4-methyl-3,4-dihydroxy cyclohexadienone 38 with a quinone methide derived at low temperature from the fluoride ion catalyzed composition of piperidinium salt 40. The resulting condensate (31) was homologated by successive esterification with protected monomeric phenol 41 to provide, after careful removal of the protecting groups, the desired thielocin A1β.

Interactions between lumophores have a critical influence on the photophysical properties of conjugated polymers. We synthesized a new series of light-harvesting polymers (poly-DSBs, I−IV) of dialkyloxy- or dialkyl-substituted distyrylbenzene (the substituents being methoxy, 2-ethylhexyloxy, and cyclohexyl) with short aliphatic linkage (methylene or ethylene) and examined the effects of interactions between lumophores and of chemical structures on the absorption, emission, and excitation spectra. The proximity between distyrylbenzene lumophores was shown to be critical to the interactions between lumophores and to the energy-transfer processes. In concentrated solutions and solid films, intermolecular aggregates exist resulting from different extents of interactions between lumophores and are found to involve at least three species:  loose, compact, and the most aligned aggregates as observed by photoluminescence and excitation spectroscopies. We also found, for the first time, sequential energy transfer from individual lumophores to the most compact, aligned aggregates via the looser intermolecular aggregates, as observed directly by time-resolved fluorescence spectroscopy. Such a process mimics energy transfer in photosynthesis units and is so efficient such that the fluorescence color can be red-shifted drastically by the presence of comparatively few aggregates and that the light evolved from concentrated solutions and films of poly-DSBs I−IV is entirely or almost the aggregation emission. Although the sequential energy-transfer process in fully conjugated electro-/photoluminescent polymers due to inhomogenity other than distributed conjugation lengths has never been directly observed at room temperature, we suggest that events similar to those observed in poly-DSBs in conjugated polymers could occur but on a much shorter time scale, i.e., a few picoseconds.

Kahalalide F, the only member of the family of peptides called kahalalides, isolated from the sacoglossan mollusc Elysia rufescens and the green alga Bryopsis sp., with important bioactivity, is in clinical trials for treatment of prostate cancer. An efficient solid-phase synthetic approach is reported. Kahalalide F presents several synthetic difficulties:  (i) an ester bond between two β-branched and sterically hindered amino acids; (ii) a didehydroamino acid; and (iii) a rather hydrophobic sequence with two fragments containing several β-branched amino acids in a row, one of them terminated with a saturated aliphatic acid. The cornerstones of our strategy were (i) a quasiorthogonal protecting system with allyl, tert-butyl, fluorenyl, and trityl-based groups, (ii) azabenzotriazole coupling reagents, (iii) formation of the didehydroamino acid residue on the solid phase, and (iv) cyclization and final purification in solution. HPLC, high-field NMR, and biological activity studies showed that the correct stereochemistry of the natural product is that proposed by Rinehart et al. whereas the stereochemistry proposed by Scheuer et al. is that of a biologically less active diastereoisomer.

Eight highly modified Daphniphyllum alkaloids with unprecedented fused hexa- or pentacyclic skeletons, daphnicyclidins A−H (1−8), have been isolated from the stems of Daphniphyllum humile and D. teijsmanni, and their structures were elucidated on the basis of spectroscopic data and chemical means. The stereochemistry was elucidated by combination of NOESY correlations, X-ray crystallographic data, and CD analyses.

A thorough analysis of the vibrational features of the titanium silicalite-1 (TS-1) catalyst is presented, based on quantitative IR measurements, Raman and resonant Raman experiments, quantitative XANES, and quantum chemical calculations on cluster and periodic models. The linear correlation of the intensity of the IR and Raman bands located at 960 and 1125 cm^-1 and the XANES peak at 4967 eV with the amount of tetrahedral Ti are quantitatively demonstrated. Raman and resonant Raman spectra of silicalite and TS-1 with variable Ti content are presented, showing main features at 960 and 1125 cm^-1 associated with titanium insertion into the zeolite framework. The enhancement of the intensity of the 1125 cm^-1 feature and the invariance of the 960 cm^-1 feature in UV−Raman experiments, are discussed in terms of resonant Raman selection rules. Quantum chemical calculations on cluster models Si[OSi(OH)₃]₄ and Ti[OSi(OH)₃]₄ at the B3LYP/6-31G(d) level of theory provide the basis for the assignment of the main vibrational contributions and for the understanding of Raman enhancement. The resonance-enhanced 1125 cm^-1 mode is unambiguously associated with a totally symmetric vibration of the TiO₄ tetrahedron, achieved through in-phase antisymmetric stretching of the four connected Ti−O−Si bridges. This vibration can also be described as a totally symmetric stretching of the four Si−O bonds pointing toward Ti. The resonance enhancement of this feature is explained in terms of the electronic structure of the Ti-containing moiety. Asymmetric stretching modes of TO₄ units show distinct behavior when (i) T is occupied by Si as in perfect silicalite, (ii) T is occupied by Ti as in TS-1, or (iii) the oxygen atom belongs to an OH group, such as in terminal tetrahedra of cluster models and in real defective zeolites. Asymmetric SiO₄ and TiO₄ stretching modes appear above and below 1000 cm^-1, respectively, when they are achieved through antisymmetric stretching of the T−O−Si bridges, and around 800 cm^-1 (in both SiO₄ and TiO₄) when they involve symmetric stretching of the T−O−Si units. In purely siliceous models, the transparency gap between the main peaks at 800 and 1100 cm^-1 contains only vibrational features associated with terminal Si−OH groups, while in Ti−containing models it contains also the above-mentioned asymmetric TiO₄ modes, which in turn are strongly coupled with Si−OH stretching modes. Calculations on periodic models of silicalite and TS-1 free of OH groups using the QMPOT embedding method correctly reproduce the transparency gap of silicalite and the appearance of asymmetric TiO₄ vibrations at 960 cm^-1 in TS-1. Finally, we demonstrate, for the first time, that the distortion of the tetrahedral symmetry around Ti caused by water adsorption quenches the UV−Raman enhancement of the 1125 cm^-1 band.

A new class of epoxy nanocomposites with completely defined organic/inorganic phases was prepared by reacting octakis(glycidyldimethylsiloxy)octasilsesquioxane [(glydicylMe₂SiOSiO_1.5)₈] (OG) with diaminodiphenylmethane (DDM) at various compositional ratios. The effects of reaction curing conditions on nanostructural organization and mechanical properties were explored. A commercial epoxy resin based on the diglycidyl ether of bisphenol A (DGEBA) was used as a reference material throughout these studies. FTIR was used to follow the curing process and to demonstrate that the silsesquioxane structure is preserved during processing. OG/DDM composites possess comparable tensile moduli (E) and fracture toughness (K_IC) to, and better thermal stabilities than, DGEBA/DDM cured under similar conditions. Dynamic mechanical analysis and model reaction studies suggest that the maximum cross-link density is obtained at N = 0.5 (NH₂:epoxy groups = 0.5) whereas the mechanical properties are maximized at N = 1.0. Digestion of the inorganic core with HF followed by GPC analysis of the resulting organic tether fragments when combined with the model reaction studies confirms that, at N = 0.5, each organic tether connects four cubes, while, at N = 1.0, linear tethers connecting two cubes dominate the network structure. Thus, well-defined nanocomposites with controlled variation of the organic tether architecture can be made and their properties assessed.

The UV−visible absorption spectra of [Ru(E)(E‘)(CO)₂(iPr-DAB)] (E = E‘ = SnPh₃ or Cl; E = SnPh₃ or Cl, E‘ = CH₃; iPr-DAB = N,N‘-di-isopropyl-1,4-diaza-1,3-butadiene) are investigated using CASSCF/CASPT2 and TD-DFT calculations on model complexes [Ru(E)(E‘)(CO)₂(Me-DAB)] (E = E‘ = SnH₃ or Cl; E = SnH₃ or Cl, E‘ = CH₃; Me-DAB = N,N‘-dimethyl-1,4-diaza-1,3-butadiene). The calculated transition energies and oscillator strengths allow an unambiguous assignment of the spectra of the nonhalide complexes [Ru(SnPh₃)₂(CO)₂(iPr-DAB)] and [Ru(SnPh₃)(Me)(CO)₂(iPr-DAB)]. The agreement between the CASSCF/CASPT2 and TD-DFT approaches is remarkably good in the case of these nonhalide complexes. The lowest-energy part of the spectrum (visible absorption) originates in electronic transitions that correspond to excitations from the axial E−Ru−E‘ σ₂ orbital into the low-lying π*_DAB orbital (σ-bond-to-ligand charge transfer, SBLCT, transitions), while the absorption between 25 000 and 35 000 cm^-1 is due to metal-to-ligand charge transfer (MLCT) excitations from the 4d_Ru orbitals to π*_DAB (MLCT). Above 35 000 cm^-1, the transitions mostly correspond to MLCT and SBLCT excitations into π*_CO orbitals. Analysis of the occupied σ orbitals involved in electronic transitions of the nonhalide complexes shows that the Kohn−Sham orbitals are generally more delocalized than their CASSCF/CASPT2 counterparts. The CASSCF/CASPT2 and TD-DFT approaches lead to different descriptions of electronic transitions of the halide complexes [Ru(Cl)₂(CO)₂(Me-DAB)] and [Ru(Cl)(Me)(CO)₂(Me-DAB)]. CASSCF/CASPT2 reproduces well the observed blue-shift of the lowest absorption band on going from the nonhalide to halide complexes. TD-DFT systematically underestimates the transition energies of these complexes, although it reproduces the general spectral features. The CASSCF/CASPT2 and TD-DFT techniques differ significantly in their assessment of the chloride contribution. Thus, CASSCF/CASPT2 assigns the lowest-energy absorption to predominantly Ru → DAB MLCT transitions, while TD-DFT predicts a mixed XLCT/MLCT character, with the XLCT component being predominant. (XLCT stands for halide (X)-to-ligand-charge transfer.) Analysis of Kohn−Sham orbitals shows a very important 3p_Cl admixture into the high-lying occupied orbitals, in contrast to the CASSCF/CASSPT2 molecular orbitals which are nearly pure 4d_Ru with the usual contribution of the back-donation to π*_CO orbitals. Further dramatic differences were found between characters of the occupied σ orbitals, as calculated by CASSCF/CASPT2 and DFT. They differ even in their bonding character with respect to the axial E−Ru and Cl−Ru bonds. These differences are attributed to a drawback of the DFT technique with respect to the dynamical correlation effects which become very important in complexes with a polar Ru−Cl bond. Similar differences in the CASSCF/CASPT2 and TD-DFT descriptions of the lowest allowed transition of [Ru(Cl)₂(CO)₂(Me-DAB)] and [Ru(Cl)(Me)(CO)₂(Me-DAB)] were found by comparing the changes of Mulliken population upon excitation. This comparison also reveals that CASSCF/CASPT2 generally predicts smaller electron density redistribution upon excitation than TD-DFT, despite the more localized character of CASSCF/CASPT2 molecular orbitals.

Sequences of guanines, GG and GGG, are known to be readily oxidized, forming radical cations, i.e., hole traps, on DNA. The trapping probability of GG is less than that of GGG. Lewis et al. (J. Am. Chem. Soc. 2000, 122, 12037) have used measurements on synthetic hairpins to determine the free energy liberated when a hole goes from the radical cation G⁺ to GG or to GGG. They find these free energies to be of the order of thermal energy at room temperature, in contradiction to the expectation by many of much greater trap depths. We have calculated the wave function of a hole on G, on GG, and on GGG surrounded by adenines, as in the Lewis et al. experiments, using a simple tight-binding model. We find that to account for the shallow traps found by them it is necessary that the difference in ionization potentials of contiguous guanine and adenine be smaller by about 0.2 eV than the 0.4 eV found for isolated bases. Using this value and taking into account polaron formation, we find the wave functions of holes trapped on G, GG, or GGG to extend over ∼6 sites (bases) and with energy level differences in good agreement with the values found by Lewis et al.

Proton translocation along ammonia wires is investigated in 7-hydroxyquinoline·(NH₃)_n clusters, both experimentally by laser spectroscopy and theoretically by Hartree−Fock and density functional (DFT) calculations. These clusters serve as realistic finite-size models for proton transfer along a chain of hydrogen-bonded solvent molecules. In the enol tautomer of 7-hydroxyquinoline (7-HQ), the OH group acts as a proton injection site into the (NH₃)_n cluster. Proton translocation along a chain of three NH₃ molecules within the cluster can take place, followed by reprotonation of 7-HQ at the quinolinic N atom, forming the 7-ketoquinoline tautomer. Exoergic proton transfer from the OH group of 7-HQ to the closest NH₃ molecule within the cluster giving a zwitterion 7-HQ^-·(NH₃)₆H⁺ (denoted PT-A) occurs at a threshold cluster size of n = 6 in the DFT calculations and at n = 5 or 6 experimentally. Three further locally stable zwitterion clusters denoted PT-B, PT-B‘, and PT-C, the keto tautomer, and several transition structures along the proton translocation path were characterized theoretically. Grotthus-type proton-hopping mechanisms occur for three of the proton transfer steps, which have low barriers and are exoergic or weakly endoergic. The step with the highest barrier involves a complex proton transfer mechanism, involving structural reorganization and large-scale diffusive motions of the cluster.

⁷Li magic angle spinning solid-state nuclear magnetic resonance is applied to investigate the lithium local environment and lithium ion mobility in tetragonal anatase TiO₂ and orthorhombic lithium titanate Li_0.6TiO₂. Upon lithium insertion, an increasing fraction of the material changes its crystallographic structure from anatase TiO₂ to lithium titanate Li_0.6TiO₂. Phase separation occurs, and as a result, the Li-rich lithium titanate phase is coexisting with the Li-poor TiO₂ phase containing only small Li amounts ≈ 0.01. In both the anatase and the lithium titanate lattice, Li is found to be hopping over the available sites with activation energies of 0.2 and 0.09 eV, respectively. This leads to rapid microscopic diffusion rates at room temperature (D_micr = 4.7 × 10^-12 cm² s^-1 in anatase and D_micr = 1.3 × 10^-11 cm² s^-1 in lithium titanate). However, macroscopic intercalation data show activation energies of ∼ 0.5 eV and smaller diffusion coefficients. We suggest that the diffusion through the phase boundary is determining the activation energy of the overall diffusion and the overall diffusion rate itself. The chemical shift of lithium in anatase is independent of temperature up to ∼250 K but decreases at higher temperatures, reflecting a change in the 3d conduction electron densities. The Li mobility becomes prominent from this same temperature showing that such electronic effects possibly facilitate the mobility.

A series of soluble hexabenzocoronene (HBC) derivatives with pendant optically active (S)-3,7-dimethyloctanyl and (R,S)-3,7-dimethyloctanyl (mixture of stereoisomers) hydrocarbon side chains with and without a phenylene spacer were assembled into differently ordered arrays at the interface between a solution and the basal plane of highly oriented pyrolytic graphite (HOPG). Molecularly resolved scanning tunneling microscopy (STM) images revealed that all derivatives self-assemble into oriented crystals in quasi-two dimensions. However, while for the alkyl-substituted HBCs (1,4) all of the single aromatic cores within a monolayer exhibit the same contrast in the STM, the single aromatic cores with a phenylene group between the alkyl side chains and the aromatic core (2a,2b,3) exhibit different contrasts within a monolayer. For the disks carrying racemic branched or n-alkyl side chains (2b,3) a random distribution of the two different contrasts within the 2D-crystal is observed, while the optically active phenylene-alkyl-substituted HBC (2a) exhibits a periodical distribution of three contrasts within the monolayer. We attribute the different contrasts of the aromatic cores in the presence of the phenylene groups to a loss of the planarity of the whole molecule and different conformations, which allow the conjugated disks to attain different equilibrium positions above the surface of HOPG. In the case of the optically active side chains a regular superstructure with three distinctly different positions such as in a staircase is attained. The self-assembly processes are governed by the interplay of intramolecular as well as intermolecular and interfacial interactions. In the present case, the interactions may induce both the molecules to acquire well distinct positions along the z axis and to adopt different conformations. The reported results open new avenues of exploration. For instance, the different couplings of conjugated molecules with the substrate at different separations can be investigated by means of scanning tunneling spectroscopy (STS). Furthermore, experiments on the STM tip-induced switching of single molecules embedded in a monolayer appear feasible.

Material transports in hyaluronan (HA) aqueous solution were investigated applying two different techniques, i.e., pulsed field gradient NMR (PFG-NMR) and photochemical quenching, to the measurement of diffusion constants to show a sharp contrast resulting from the difference of the spectroscopic observation time while the same probe molecules were commonly used in two experiments. The value from PFG-NMR reflects the relatively long transport along which the majority of the molecules are retarded by the mesh structure of HA solution. In such inhomogeneous fluids, the observable diffusion constant should generally depend on the observation time and, i.e., the averaged distance of diffusion. Quantitative discussion, which compares the obtained characteristic distance of diffusion with the pore size, clarifies the role of the nano-microstructure of HA solution forming small pores surrounded by the polymer chain networks.

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt−rate profiles:  k_cat/K_M = (k_cat/K_M)_MAX/[1 + ([Na⁺]/K_Na⁺)^n‘], where (k_cat/K_M)_MAX is the physical limit of k_cat/K_M, K_Na⁺ is the salt concentration at which k_cat/K_M = (k_cat/K_M)_MAX/2, and −n‘ is the slope of the linear region in a plot of log(k_cat/K_M) versus log [Na⁺]. The value of n‘ is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k_cat/K_M values that can exceed 10⁹ M^-1 s^-1. Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n‘. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n‘. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n‘, as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt−rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.