Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.     

Interferometric coherence transfer modulations in triply vibrationally enhanced four-wave mixing

Triply vibrationally enhanced (TRIVE) four-wave mixing (FWM) spectroscopy in a mixed frequency/time domain experiment contains new output coherences that isolate nonlinear pathways that involve coherence transfer. Coherence transfer occurs when a thermal bath induces coupling between two states so a quantum mechanical entanglement of a pair of quantum states evolves to entangle a new pair of quantum states. The FWM includes several equivalent coherence pathways that interfere and create a temporal modulation of the output coherence that is a signature of coherence transfer. The transfer shifts the output coherence frequency and isolates coherence transfer pathways from the stronger FWM processes that form the basis of coherent multidimensional spectroscopy. The use of coherence transfer offers the opportunity for another form of coherent multidimensional spectroscopy where cross-peaks appear because of the coherence transfer between quantum states. Since this approach is based on frequency domain methods, it requires only short-term phase coherence during the excitation process so the method is not constrained to accessing the quantum states lying within the excitation pulse bandwidth.     

Using switched angle spinning to simplify NMR spectra of strongly oriented samples

This contribution describes a method that manipulates the alignment director of a liquid crystalline sample to obtain anisotropic magnetic interaction parameters, such as dipolar coupling, in an oriented liquid crystalline sample. By changing the axis of rotation with respect to the applied magnetic field in a spinning liquid crystalline sample, the dipolar couplings present in a normally complex strong coupling spectrum are scaled to a simple weak coupling spectrum. This simplified weak coupling spectrum is then correlated with the isotropic chemical shift in a switched angle spinning (SAS) two-dimensional (2D) experiment. This dipolar-isotropic 2D correlation was also observed for the case where the couplings are scaled to a degree where the spectrum approaches strong coupling. The SAS 2D correlation of C(6)F(5)Cl in the nematic liquid crystal I52 was obtained by first evolving at an angle close to the magic angle (54.7 degrees ) and then directly detecting at the magic angle. The SAS method provides a 2D correlation where the weak coupling pairs are revealed as cross-peaks in the indirect dimension separated by the isotropic chemical shifts in the direct dimension. Additionally, by using a more complex SAS method which involves three changes of the spinning axis, the solidlike spinning sideband patterns were correlated with the isotropic chemical shifts in a 2D experiment. These techniques are expected to enhance the interpretation and assignment of anisotropic magnetic interactions including dipolar couplings for molecules dissolved in oriented liquid crystalline phases.     

Spin-state selective carbon-detected HNCO with TROSY optimization in all dimensions and double echo-antiecho sensitivity enhancement in both indirect dimensions

A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of square root of 2 in both indirect dimensions and is generally applicable to many multidimensional experiments. A carbon-detected HNCO experiment, c-HNCO, without TROSY optimization and sensitivity enhancement is also designed for comparison purposes. Relaxation simulations show that for a protein with a rotational correlation time of 10 ns or larger, the c-TROSY-HNCO experiment displays comparable or higher signal-to-noise (S/N) ratios than the c-HNCO experiment, although the former selects only 1/4 of the initial magnetization relative to the later. The high resolution afforded in the directly detected carbon dimension allows direct measurement of the doublet splitting to extract 1JCalphaC' scalar and 1DCalphaC' residual dipolar couplings. Simulations indicate that the c-TROSY-HNCO experiment offers higher precision (lower uncertainty) compared to the c-HNCO experiment for larger proteins. The experiments are applied to 15N/13C/2H/[Leu,Val]-methyl-protonated IIBMannose, a protein of molecular mass 18.6 kDa with a correlation time of approximately 10 ns at 30 degrees C. The experimental pairwise root-mean-square deviation for the measured 1JCalphaC' couplings obtained from duplicate experiments is 0.77 Hz. By directly measuring the doublet splitting, the experiments described here are expected to be much more tolerant to nonuniform values of 1JCalphaC' (or 1JCalphaC' + 1DCalphaC' for aligned samples) and pulse imperfections due to the smaller number of applied pulses in the "out-and-stay" coherence transfer in the c-HNCO-TROSY experiment relative to conventional 1H-detected "out-and-back" quantitative J correlation experiments. A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of in both indirect dimensions and is generally applicable to many multidimensional experiments.     

Two-dimensional infrared spectroscopy of antiparallel beta-sheet secondary structure

We investigate the sensitivity of femtosecond Fourier transform two-dimensional infrared spectroscopy to protein secondary structure with a study of antiparallel beta-sheets. The results show that 2D IR spectroscopy is more sensitive to structural differences between proteins than traditional infrared spectroscopy, providing an observable that allows comparison to quantitative models of protein vibrational spectroscopy. 2D IR correlation spectra of the amide I region of poly-l-lysine, concanavalin A, ribonuclease A, and lysozyme show cross-peaks between the IR-active transitions that are characteristic of amide I couplings for polypeptides in antiparallel hydrogen-bonding registry. For poly-l-lysine, the 2D IR spectrum contains the eight-peak structure expected for two dominant vibrations of an extended, ordered antiparallel beta-sheet. In the proteins with antiparallel beta-sheets, interference effects between the diagonal and cross-peaks arising from the sheets, combined with diagonally elongated resonances from additional amide transitions, lead to a characteristic "Z"-shaped pattern for the amide I region in the 2D IR spectrum. We discuss in detail how the number of strands in the sheet, the local configurational disorder in the sheet, the delocalization of the vibrational excitation, and the angle between transition dipole moments affect the position, splitting, amplitude, and line shape of the cross-peaks and diagonal peaks.     

Constraints on supramolecular structure in amyloid fibrils from two-dimensional solid-state NMR spectroscopy with uniform isotopic labeling

We show that strong constraints on supramolecular structure in amyloid fibrils can be obtained from solid-state nuclear magnetic resonance measurements on samples with uniformly 13C-labeled segments. The measurements exploit two-dimensional (2D) 13C-13C exchange spectroscopy in conjunction with high-speed magic angle spinning, with proton-mediated exchange of 13C nuclear spin magnetization as recently demonstrated by Baldus and co-workers (J. Am. Chem. Soc. 2002, 124, 9704-9705). Proton-mediated 2D exchange spectra of fibrils formed by residues 16-22 of the 40-residue Alzheimer's beta-amyloid peptide show strong nonsequential, intermolecular cross-peaks between alpha-carbons that dictate an antiparallel beta-sheet structure in which residue 16+k aligns with residue 22-k. The strong alpha/alpha cross-peaks are absent from conventional, direct 2D exchange spectra. Proton-mediated 2D exchange spectra of fibrils formed by residues 11-25 indicate an antiparallel beta-sheet structure with a pH-dependent intermolecular alignment. In contrast, proton-mediated 2D exchange spectra of fibrils formed by the full-length beta-amyloid peptide are consistent with a parallel beta-sheet structure. These data show that the supramolecular structure of amyloid fibrils is not determined by the amino acid sequence at the level of 7-residue or 15-residue segments. The proton-mediated 2D exchange spectra additionally demonstrate that the intermolecular alignment in the beta-sheets of these amyloid fibrils is highly ordered, with no detectable evidence for "misalignment" defects.     

A multivariate chemometric approach to fluorescence spectroscopy

A multivariate approach to the solution of problems often encountered in the spectrofluorometry of natural samples, utilising information from whole spectra is presented. (a) Piecewise direct standardisation is implemented and employed to transfer emission spectra measured with two different xenon lamps of different ages as if the spectra were measured with the same lamp. (b) It has been shown using a multivariate analysis approach that it is possible to use the raw data points instead of the smoothed data based on an algorithm included in the instrument software by the manufacturer. (c) It is documented that Raman scattering does not hamper the performance of multivariate calibration; on the contrary, in an experiment with sugar samples the concentration prediction errors become about five times lower by including the whole emission spectrum in the analysis instead of using a univariate calibration based on an emission wavelength that only reflects the analyte of interest. (d) An algorithm for variable selection is implemented and employed in the selection of optimal excitation wavelengths. Among 13 emission spectra recorded for a sugar sample at different excitation wavelengths, four of these are chosen that describe 98.51% of the total variance in the original data. (e) Finally the combination of fluorescence spectroscopy and multivariate calibration with conventional chemical data according to the near-infrared black box model is presented. The refined sugar quality parameter, the ash content and the fluorescence emission spectra are correlated by a partial least-squares regression model. Five experiments employing different monochromator slit widths and sugar concentrations are performed, and the best correlation obtained by full cross-validation of the 15 sugar samples is R = 0.98.     

Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy

Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination positions accounts for the EXSY cross-peaks present in the spectra of all adducts.     

D-HMBC versus LR-HSQMBC: Which experiment provides theoretically and experimentally the best results?

The long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) experiment is the experiment of choice for visualizing heteronuclear long-range coupling interactions ⁿJ_CH across 4–6-bonds and is experimentally superior to the decoupled heteronuclear multiple-bond correlation (D-HMBC) experiment. Yet, the exact reasons have not been fully understood and established. On the basis of our recent investigation of the nonrefocused variants LR-HSQC and HMBC, we have extended a J_HH′-dedicated investigation to the D-HMBC and LR-HSQMBC experiments. Unlike the nonrefocused variants, the influence of homonuclear couplings J_HH′ on the intensity of long-range ⁿJ_CH cross-peaks is not easily predictable and may be summarized as follows: (a) irrespective of the magnitude and number of J_HH′ interactions long-range ⁿJ_CH cross-peaks are more intense in D-HMBC spectra as long as the evolution delay Δ is not too large, because in contrast to LR-HSQMBC no J_HH′-caused intensity zeroes will occur. (b) If J_HH′ is small and Δ large, the intensity of cross peaks in D-HMBC spectra may be weakened or may even vanish at Δ = (0.25+0.5k)/J_HH′, whereas for the LR-HSQMBC this unwanted effect occurs at Δ = k + 0.5/J_HH′. Consequently, when Δ is adjusted to visualize weak ⁿJ_CH long-range correlations, our findings corroborate that there are potentially more cross-peaks expected to show up in a LR-HSQMBC spectrum compared with a D-HMBC spectrum. This has been indeed noticed experimentally, even though the intensity of a many long-range ⁿJ_CH cross-peaks may still be higher in the spectra of the D-HMBC experiment correspondingly adjusted for detecting weak ⁿJ_CH correlations.     

Solution state structure determination of silicate oligomers by 29SI NMR spectroscopy and molecular modeling

Evidence for nine new solution state silicate oligomers has been discovered by (29)Si NMR homonuclear correlation experiments of (29)Si-enriched samples. In addition to enhancing signal sensitivity, the isotopic enrichment increases the probability of the (29)Si-(29)Si two-bond scalar couplings that are necessary for the observation of internuclear correlations in 2-D experiments. The proposed assignments are validated by comparisons of experimental and simulated cross-peaks obtained with high digital resolution. The internuclear connectivity indicated by the NMR data suggests that several of these oligomers can have multiple stereoisomers, including conformers and/or diastereomers. The stabilities of these oligomers and their possible stereoisomers have been investigated by electronic structure calculations.     

Protein backbone dynamics through 13C'-13Calpha cross-relaxation in NMR spectroscopy

Internal dynamics of proteins are usually characterized by the analysis of (15)N relaxation rates that reflect the motions of NH(N) vectors. It was suggested a decade ago that additional information on backbone motions can be obtained by measuring cross-relaxation rates associated with intra-residue C'C(alpha) vectors. Here we propose a new approach to such measurements, based on the observation of the transfer between two-spin orders 2N(z)() and 2N(z)(). This amounts to "anchoring" the and operators to the N(z)() term from the amide of the next residue. In combination with symmetrical reconversion, this method greatly reduces various artifacts. The experiment is carried out on human ubiquitin at 284.1 K, where the correlation time is 7.1 ns. The motions of the C'C(alpha) vector appear more restricted than those of the NH(N) vector.     

Determination of (3)J((1)H3'-(31)P) couplings in a DNA oligomer with enhanced sensitivity employing a constant-time TOCSY difference experiment

A constant-time TOCSY difference experiment for the determination of (3)J((1)H3'-(31)P) coupling constants in non-isotope-labelled DNA oligonucleotides is presented. The method is tested on a DNA octamer and compared with the established constant-time NOESY difference method. Each (3)J((1)H3'-(31)P) coupling constant is determined from amplitude changes caused by phosphorous decoupling, which are observable on multiple cross-peaks, thus leading to a high accuracy of the value of the (3)J((1)H3'-(31)P) coupling constant. The new experiment delivers up to three times the sensitivity compared with previously reported methods.     

The importance of including local correlation times in the calculation of inter-proton distances from NMR measurements: ignoring local correlation times leads to significant errors in the conformational analysis of the Glc alpha1-2Glc alpha linkage by NMR spectroscopy

The experimental determination of oligosaccharide conformations has traditionally used cross-linkage 1H-1H NOE/ROEs. As relatively few NOEs are observed, to provide sufficient conformational constraints this method relies on: accurate quantification of NOE intensities (positive constraints); analysis of absent NOEs (negative constraints); and hence calculation of inter-proton distances using the two-spin approximation. We have compared the results obtained by using 1H 2D NOESY, ROESY and T-ROESY experiments at 500 and 700 MHz to determine the conformation of the terminal Glc alpha1-2Glc alpha linkage in a dodecasaccharide and a related tetrasaccharide. For the tetrasaccharide, the NOESY and ROESY spectra produced the same qualitative pattern of linkage cross-peaks but the quantitative pattern, the relative peak intensities, was different. For the dodecasaccharide, the NOESY and ROESY spectra at 500 MHz produced a different qualitative pattern of linkage cross-peaks, with fewer peaks in the NOESY spectrum. At 700 MHz, the NOESY and ROESY spectra of the dodecasaccharide produced the same qualitative pattern of peaks, but again the relative peak intensities were different. These differences are due to very significant differences in the local correlation times for different proton pairs across this glycosidic linkage. The local correlation time for each proton pair was measured using the ratio of the NOESY and T-ROESY cross-relaxation rates, leaving the NOESY and ROESY as independent data sets for calculating the inter-proton distances. The inter-proton distances calculated including the effects of differences in local correlation times give much more consistent results.     

Intramolecular energy transfer through charge transfer state in lanthanide compounds: a theoretical approach

A theoretical approach for the intramolecular energy transfer process involving the ligand-to-metal charge transfer (LMCT) state in lanthanide compounds is developed. Considering a two-electron interaction, both the direct Coulomb and exchange interactions are taken into account, leading to expressions from which selection rules may be derived and transfer rates may be calculated. These selection rules show that the direct Coulomb and exchange mechanisms are complementary, in the same way as obtained in previous works for the case of ligand-lanthanide ion energy transfer processes. An important result from numerical estimates is that the channel ligand-LMCT state is by far the dominant case, leading to transfer rates higher than for the channel lanthanide ion-LMCT state by several orders of magnitude. The analysis of the emission quantum yield as a function of the relative energy position of the LMCT state in a typical Eu(3+) compound allows the identification of two quenching regions, the most pronounced one occurring close to the lower ligand triplet level.     

Two-dimensional nuclear Overhauser enhancement NMR experiments on pelargonidin-3-glucopyranoside, an anthocyanin of low molecular mass.

Pelargonidin-3-glucoside has been isolated from the acidified methanolic extract of strawberries (Fragaria anannassa variety Corona) by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The pigment in acidified methanolic solution was studied by means of the two-dimensional nuclear Overhauser enhancement NMR technique, and the sugar unit was found to be attached to the 3-position on the aglycone. At +20 degrees C the pigment was found to be in the extreme narrowing limit where the NOESY cross-peaks are negative. However, at -20 degrees C this low-mass anthocyanin could be studied in the slow motion regime where the NOESY cross-peaks are positive. With a mixing time of 0.3 s, the glucose H1"-H4" proton pair was measured in the initial cross-relaxation rate and their cross-peak volume corresponded to the H1"-H4" distance found in a 4C1 chair conformation.     

Affinity NMR: decoding DNA binding

We have shown that affinity NMR can be used to edit a NMR spectrum so that ligands that have affinity to DNA can be observed in the presence of other nonbinding molecules. Diffusion encoded spectroscopy (DECODES) can be used to identify the binding ligands. We were able to identify Hoechst 33342 as binding to the Drew-Dickerson dodecamer d(CGCGAATTCGCG)2 in the presence of the nonbinding molecules adenine, adenosine, and thiamine. Affinity NMR appears to be readily applicable to DNA systems for the following reasons. (1) The relaxation rate of the DNA oligonucleotides is favorable, thus the signal intensity loss due to relaxation is not severe. (2) A comparison of the patterns of the DNA cross-peaks upon binding in the two-dimensional total correlation spectroscopy and correlation spectroscopy spectrum are easily performed, and the ligand signals in the two-dimensional DECODES spectrum can be readily identified. (3) The aromatic part of the DNA spectrum is devoid of 2D cross-peaks in these correlation spectra, greatly facilitating the interpretation of the bound ligand in the DECODES spectrum.     

Kinetics of RNA refolding in dynamic equilibrium by 1H-detected 15N exchange NMR spectroscopy

By implementing new NMR methods that were designed to map very slow exchange processes we have investigated and characterized the refolding kinetics of a thermodynamically stable 34mer RNA sequence in dynamic equilibrium. The RNA sequence was designed to undergo a topologically favored conformational exchange between different hairpin folds, serving as a model to estimate the minimal time required for more complex RNA folding processes. Chemically prepared RNA sequences with sequence-selective (15)N labels provided the required signal separation and allowed a straightforward signal assignment of the imino protons by HNN correlation experiments. The 2D version of the new (1)H-detected (15)N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of (15)N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau(obs) approximately T(1){(15)Nu} < 5 s) and a third fold which is static on this time scale. A 1D version of the (15)N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies E(a) and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies are comparable to values obtained for the slow refolding of RNA sequences of similar thermodynamic stability but less favorable topology.     

Multiresonant coherent multidimensional vibrational spectroscopy of aromatic systems: pyridine, a model system

Multiresonant four wave mixing has been used to measure the coherent multidimensional spectroscopy (CMDS) of representative aromatic ring modes using pyridine as a model system. This work identifies the cross-peaks that appear between several modes and measures their coherent and incoherent dynamics. The work also explores the consequences of using multiresonant CMDS for molecules with transition moments that are typical of most vibrational modes. Typically, CMDS experiments rely on using transitions with exceptionally large transition moments. To observe cross-peaks, the pyridine concentration was raised until absorption effects became very important. These effects interfere with the parametric CMDS coherence pathways, but they do not make important contributions to the nonparametric pathways.     

Characterization of a chiral stationary phase by HR/MAS NMR spectroscopy and investigation of enantioselective interaction with chiral ligates by transferred NOE

The surface chemistry of a chiral stationary phase (CSP) with a (tert-butyl carbamoyl) quinine selector immobilized on thiol-modified silica has been characterized by (1)H HR/MAS NMR and (29)Si CP/MAS NMR spectroscopy. The mostly well-resolved (1)H signals could be assigned to stem from the surface-bound selector and the latter suggested a bi- and trifunctional silane linkage. Suspended-state NMR spectroscopy thus proved a well-characterized surface chemistry as proposed. To study chiral recognition phenomena in the presence of the CSP, (1)H HR/MAS 2D transfer NOESY investigations in methanol-d(4) have been undertaken with various solutes including N-3,5-dinitrobenzoyl derivatives of leucine (DNB-Leu) and N-acetyl phenylalanine (Ac-Phe). Both (R)- and (S)-enantiomers of DNB-Leu and Ac-Phe interacted with the tBuCQN-CSP as indicated by negative cross-peaks in the trNOESY spectra, while the 2D NOESY of the dissolved solutes in absence of the chiral stationary phase showed positive cross-peaks. The intensities of the trNOE cross-peaks were much stronger for the (S)-enantiomers. This stereoselectivity paralleled the experimental chromatographic behavior, where the (S)-enantiomers revealed stronger binding and retention on the tBuCQN-CSP as well. Hence, we were able to correlate the retention behavior to the trNOE NMR spectroscopic data in a qualitative manner.     

Photo-induced intermolecular electron transfer from electron donating solvents to Coumarin dyes in bile salt aggregates: role of diffusion in electron transfer reaction

The photo-induced electron transfer between Coumarin dyes and aromatic amines has been investigated using steady state and time-resolved fluorescence quenching studies. We have observed a Marcus type inversion in the electron transfer rate in correlation of quenching constant to the free energy change occurred during reaction. To justify the "inverted region" obtained in the correlation of quenching constant versus free energy curve, we have performed anisotropy measurement and estimated the several diffusional parameters. The translational diffusion coefficients exhibit a similar picture like electron transfer rate constant when it is plotted against free energy. Thus we argued that the diffusion has played an important role in the electron transfer kinetics.