Two-dimensional Correlation Spectra Research Articles

Two-Dimensional Correlation Analysis, Fourier Self-Deconvolution, and Second-Derivative Studies on Assignments of the CH2 Stretching Bands for Cast Films of 2-Dodecyl-, 2-Pentadecyl-, and 2-Octadecyl-7,7,8,8-Tetracyanoquinodimethane

Infrared spectra have been measured for cast films of three kinds of 2-alkyl-7,7,8,8-tetracyanoquinodimethanes (C nTCNQ) prepared on KBr plates. Each infrared spectrum shows two components for the CH2 antisymmetric stretching band (2926 and 2918 cm−1), and the relative intensity of the two bands changes with the length of the alkyl chain. In contrast to the characteristic doublet band in the antisymmetric stretching band region, only one band seems to appear near 2850 cm−1 in the corresponding CH2 symmetric stretching band region. In order to reveal the number of the bands appearing in the CH2 antisymmetric and symmetric stretching regions and to explore the origins of these bands, Fourier self-deconvolution (FSD) and second-derivative methods have been applied to the infrared spectra of the cast films. The FSD analysis of the spectra for the three kinds of C nTCNQ derivatives measured at a 4 cm−1 resolution demonstrates that the CH2 symmetric stretching band also consists of two components (near 2853 and 2848 cm−1). The same conclusion has been obtained by the second derivative of the spectra measured at 1 cm−1 resolution. An asynchronous two-dimensional (2D) correlation spectrum created from time-dependent infrared spectra for a one-layer Langmuir–Blodgett (LB) film develops cross peaks at (2927 and 2916 cm−1) and (2854 and 2846 cm−1), suggesting that the bands at 2927 and 2916 cm−1 come from different species and those at 2854 and 2846 cm−1 also have different origins. The bands at 2918 and 2848 cm−1 may be due to the interdigitated part of the alkyl chain in the cast film, which has ordered ( trans-zigzag) form, while the bands near 2926 and 2853 cm−1 are probably ascribed to the noninterdigitated part of the alkyl chain, which has disordered form with several gauche conformations.

The pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases from wheat bran of Triticum aestivum L. cv. Nourin #61.

Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate (InsP6). The pathway of hydrolysis of InsP6 by phytase from wheat bran of Triticum aestivum L. cv. Nourin #61 is proved in this study. Structures of the intermediates were established by a variety of nuclear magnetic resonance techniques (1H-, two-dimensional 1H-1H coupling-correlation spectra and two-dimensional 31P-1H correlation spectra), gas chromatography, and bioassay. On the basis of the structures identified, initial hydrolysis of the phosphate ester occurs at the D/L-4 position of InsP6 to yield D/L-Ins (1, 2, 3, 5, 6) P5. After the dephosphorylation, the pathway of dephosphorylation is divided into two routes. The main route proceeds via D/L-Ins (1, 2, 5, 6) P4, D/L-Ins (1, 2, 6) P3 and D/L-Ins (1, 2) P2, while the minor route proceeds via D/L-Ins (1, 2, 3, 6) P4, Ins (1, 2, 3) P3 and D/L-Ins (1, 2) P2. D/L-Ins (1, 2) P2 is hydrolyzed at the D/L-1 or 2-position, and finally myo-inositol is produced.

Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state.

We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-13C,15N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical 13C peak line widths are 0.5 ppm, resulting in Calpha-Cbeta and Calpha-CO regions that exhibit many resolved peaks. Two-dimensional carbon-carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 micromol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings.

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between 1HN-13Calpha, 15N-13Calpha, 1HN-13Calpha(i-1), 15N-13Calpha(i-1), 1HN-13C'(i-1), 15N-13C'(i-1) and 13C'(i-1)-13Calpha(i-1) without introducing nonessential spectral crowding compared with an ordinary two-dimensional 15N-1H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of alpha/beta-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure-activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.

Neu5Acalpha3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes.

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N-acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion-exchange chromatography. Three subfractions were deacetylated, analysed by matrix-assisted laser desorption ionization-time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two-dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acalpha3Galbeta4GlcNAc, and Sambucus nigra, specific for Neu5Acalpha6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3-linked, rather than 6-linked, sialic acid is essential for binding.

Analysis of Near-Infrared Spectra of Complicated Biological Fluids by Two-Dimensional Correlation Spectroscopy: Protein and Fat Concentration-Dependent Spectral Changes of Milk

Generalized two-dimensional (2D) correlation spectroscopy has been applied to analyze near-infrared (NIR) spectra of milk with different protein and fat concentrations. The NIR spectra of milk show rather poor signal-to-noise ratios compared with those of a protein or fat solution and have changing baselines from one spectrum to another. Poor signal-to-noise ratio and variations in baseline are common problems for NIR spectra of real-world samples. This study aims at expanding the utility of generalized 2D correlation spectroscopy to complicated multicomponent biological systems. In order to overcome the above two problems, we have employed multiplicative scatter correction (MSC) and smoothing as pretreatment procedures of the milk spectra selected for the calculation of 2D NIR correlation. 2D synchronous correlation spectra in the 2000–2400 nm region constructed from protein or fat concentration-dependent spectral changes of milk sharply enhance bands assignable to proteins or fats, respectively. It has been found that a power spectrum along the diagonal line in a synchronous spectrum very effectively shows the contribution of a particular component to the NIR spectra of milk. In fact, for example, the power spectrum for the fat concentration-dependent spectral changes of milk is very close to an NIR spectrum of fat itself. Two-dimensional asynchronous correlation spectra demonstrate the existence of bands that cannot be identified even by calculation of second derivatives and chemometrics analysis of the spectra. The asynchronous spectra also elucidate interaction between fats, proteins, and water.

Two-Dimensional Correlation Spectroscopy: Effect of Normalization of the Dynamic Spectra

Simulation studies have demonstrated that linear nonselective intensity variations coupled with nonlinear selective intensity changes may develop new features in two-dimensional (2D) correlation spectra. Some types of linear nonselective intensity changes are hardly seen in the normal and synchronous 2D correlation spectrum. In contrast, they may develop quite strong features in the companion asynchronous spectrum, especially if the selective intensity variations for different bands have similar response functions. The simplest way of removing this effect is normalization of the dynamic spectra prior to 2D correlation analysis. This operation can be easily performed if we know the relationship between the perturbation and the nonspecific intensity variations. Otherwise, one has to employ an “internal reference” for normalization of the experimental spectra. The “internal reference” means the band that does not selectively change in its intensity under given perturbation. Fourier transform near-infrared (FT-NIR) measurements of octan-1-ol in CCl4 revealed a strong correlation between the concentration and the integrated intensity of the second overtone of the v(C–H) band. Also a strong correlation was found between the integrated intensity of the same band and the temperature-induced density changes of pure octan-1-ol. Thus, in the NIR region the second overtone of the v(C–H) band can be successfully applied for normalization of both the concentration and temperature-perturbed spectra of numerous organic samples. The most complicated situation appears for a rheo-optical experiment involving pronounced deformation, where any simple normalization of the experimental spectra cannot be applied. In this instance, knowledge of the exact relationship between the strain and the sample thickness is required.

Measurement of 1JNC′ and 2JHNC′ Couplings from Spin-State-Selective Two-Dimensional Correlation Spectrum

A method for the measurement of 1JNC′ and 2JHNC′ coupling constants from a simplified two-dimensional [15N, 1H] correlation spectrum is presented. The multiplet components of the 1JNC′ doublet in the indirect dimension and 2JHNC′ in the direct dimension are separated into two subspectra by spin-state-selective filters. Thus each subspectrum contains no more peaks than the conventional [15N, 1H]-HSQC spectrum. Furthermore, the method for the measurement of 1JNC′ and 2JHNC′ is designed to exploit destructive relaxation interference (TROSY). The results are verified against the measurements of 1JNC′ from spin-state-selective [13C′, 1H] correlation spectra recorded with additional sequence described here.

Generalized Two-Dimensional Fourier Transform Infrared Multiple-Angle Attenuated Total Reflection Spectral Depth-Profiling Analysis of Multilayer Samples

This paper reports the application of a generalized two-dimensional (G2D) correlation method in multiple-angle single-reflection attenuated total reflection (ATR) spectral depth-profiling analysis of multilayer samples. In this method, ATR spectra at different incident angles are used to generate two-dimensional (2D) correlation spectra. The relative spatial interactions of correlated signals are then determined from the signs of the corresponding contours of the asynchronous map. In order to apply this method to multilayer samples, effects of dispersion in refractive indices of materials which create pseudo-contours were first investigated with simulated ATR spectra. Model ATR spectra were generated from conventional antisymmetric linear combinations of the Lorentzian functions. The use of Ge ATR crystals minimizes the pseudo-contours and allows the method to be successfully applied to actual multilayer samples. A 0.1 μm thick gelatin layer on a polyester film and a 0.2 μm thick polycarbonate layer on the gelatin/polyester film were investigated as actual model samples. In the model samples, although the layers were thin, the relative depth of each layer was easily determined.

Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1.

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP. The two-dimensional (2D) 1H-15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by alpha-chymotrypsin yielded a approximately 9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1CTD. As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined. As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix. Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1CTD. MBF1CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.

Hydrogen−Deuterium Exchange of Streptavidin and Its Complex with Biotin Studied by 2D-Attenuated Total Reflection Fourier Transform Infrared Spectroscopy

Hydrogen−deuterium exchange for streptavidin and its complex with biotin is studied by means of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. To analyze the spectral changes upon deuteration, difference spectra and two-dimensional correlation spectra are calculated. We find that the exchange rate varies with the secondary structure in which the exchanging amide protons are incorporated. The most slowly exchanging protons, with a characteristic time constant on the order of hours, are part of the β-sheet secondary structure. The separation in time of exchange of the β-sheet from other structural elements allows the amide II and II‘ frequencies of the β-sheet (1530 and 1445 cm-1) to be identified. A second component which exchanges more rapidly than the β-sheet is characterized by its amide I frequencies 1680, 1640, and 1465 cm-1. This component is attributed to the exchange of amide groups in secondary structures other than the central β-barrel. Binding of the ligand res...

Site Connectivities in Amorphous Materials Studied by Double-Resonance NMR of Quadrupolar Nuclei: High-Resolution 11B ↔ 27Al Spectroscopy of Aluminoborate Glasses

The utility of heteronuclear double-resonance NMR methods involving the quadrupolar nuclei 11B and 27Al is demonstrated for probing the intermediate-range order in aluminoborate glasses. Results from both rotational echo double resonance (REDOR) and heteronuclear cross-polarization magic angle spinning (CPMAS) studies are presented and discussed. While both techniques are strongly affected by the presence of large quadrupolar splittings, a working strategy is developed on the basis of which reliable structural interpretations are possible. Using this strategy, the effects of the cation type M on the structures of two basic glass compositions, 25M(2)O-45B2O3-30Al2O3 and 40M(2)O-40B2O3-20Al2O3 (M = Na, Ca, Mg), are discussed. Within each series having the same basic network composition, replacement of the modifier cation Na by Ca or Mg produces a large change in the speciation of framework aluminum (“cation effect”): while in the sodium-containing glass the overwhelming fraction of Al is present as tetrahedral AlO4/2 sites, the calcium- and magnesium-containing glasses also possess larger fractions of five- and six-coordinated aluminum sites. 27Al{11B} REDOR results indicate that all of these aluminum environments interact equally strongly with the dominant BO3/2 sites in both glasses. In addition, the reverse 11B{27Al} REDOR results reveal that the extent of the AlO4/2−BO4/2 interaction in the network is highly dependent on the framework composition and on the M cation type. While sodium aluminoborate glasses reveal strong chemical ordering (avoidance of intertetrahedral linkages), the analogous Mg2+-based glasses tend toward a randomization of connectivities. Finally, two-dimensional heteronuclear correlation spectra obtained via 11B{27Al} CPMAS suggest that AlO5 and AlO6 sites can provide a mechanism for stabilizing tetrahedral BO4/2 units.

Crystallographic and NMR analysis of 9-phenylthiophenanthrene and 9-tert-butylthiophenanthrene

The crystal structures of 9-phenylthiophenanthrene (C20H14S) and 9-tert-butylthiophenanthrene (C18H18S) were determined at 193 K. The former crystallizes in orthorhombic space group P212121 (No. 19) with cell dimensions a = 5.602(2), b = 9.247(3), and c = 27.508(12) Å and the latter in orthorhombic space group Pbcn (No. 60) with cell parameters a = 21.335(4), b = 7.540(2), and c = 18.197(4) Å. In both compounds the substituents at sulfur are nearly perpendicular to the phenanthrene plane. NMR spectra of the compounds were recorded in CDCl3. The 1H NMR spectral parameters were analyzed in detail by using an iterative spectral analysis program. The 13C{1H} resonances were fully assigned with the aid of two-dimensional heteronuclear chemical shift correlation spectra. On the basis of the NOE difference spectra the orientation of the side chain was concluded to be similar to that in the solid state.

Interpretation of Two-Dimensional Correlation Spectra: Science or Art?

It has been shown that the most common perturbations of conventional (one-dimensional) spectra such as random noise, baseline fluctuations, band position, and width changes may complicate two-dimensional (2D) correlation spectra, sometimes making them completely useless. In addition, two different physical causes may generate similar patterns for the synchronous and asynchronous spectra. Some of these effects, such as random noise and baseline fluctuations, can be eliminated from the input data, and one can recover the original appearance of 2D correlation spectra. The other effects, such as the frequency shift and bandwidth variation, cannot be removed from the experimental spectra. In this instance, the number and position of the correlation peaks can be elucidated by simulation studies. This report presents a few examples of typical patterns found in the synchronous and asynchronous spectra affected by those perturbations. Long streaks in 2D correlation spectra reveal extensive baseline fluctuations in the original data set. A simple offset often significantly reduces the extent of this effect. When no reasonable baseline correlations can be performed, the second derivative may solve this problem. In most cases, the perturbationaveraged spectrum is recommended as a reference. However, it has been proved that the calculation of 2D correlation spectra without any reference spectrum may also provide useful information, especially for data heavily influenced by noise or baseline fluctuations. In the majority of real-world systems, the spectral changes are a continuous function of applied perturbation. Thus, 2D correlation spectra yield information about the relative rate of intensity variations rather than the sequence of spectral events.

A Study of the Coordination Geometry of the Tetrapyridine Copper(II) Complex in NaY and MCM-41 by Two-Dimensional Electron Spin Resonance HYSCORE Spectroscopy

The coordination geometry of the tetrapyridine copper(II) complex in zeolite Cu−NaY and mesoporous siliceous MCM-41 material is investigated by two-dimensional proton and deuterium hyperfine sublevel correlation (HYSCORE) spectroscopy. Different orientations of the pyridine ligands with respect to the complex plane of the tetrapyridine copper(II) complex were found in the two materials. The ligands are predominately arranged with their molecular plane perpendicular to the complex plane in zeolite Cu−NaY. Otherwise, in Cu−MCM-41 materials the ligands have a mean orientation of their molecular plane parallel to the complex plane. However, the two-dimensional hyperfine sublevel correlation spectra reveal severe distortions of the overall complex. The different coordination geometries of the tetrapyridine copper(II) complex in both materials result from the interaction of the complex with the inner surface of each molecular sieve. Consequently, much severe distortion of the complex geometry was found in the mesoporous Cu−MCM-41 material with its amorphous wall structure in comparison to crystalline Y zeolite. The incorporation of the tetrapyridine Cu(II) complex into Y zeolite also alters the electron spin density distribution within the complex. The results indicate a substantial influence of the framework of the Y zeolite on the unpaired electron of the encapsulated complex.

Cation sites in mixed-alkali phosphate glasses

Two lithium/sodium mixed-alkali phosphate glasses were investigated with solid-state two-dimensional heteronuclear correlation NMR. The systems represent two distinct families of mixed-alkali glasses, as they contain a fixed sodium-to-lithium ratio of 1:3, but different overall concentrations of cations. In both glasses, direct NMR evidence is found for the presence of multiple sodium cation environments using frequency-selective editing of two-dimensional correlation spectra. Frequency-specific subspectra were obtained by projection of 23Na resonances correlated to distinct phosphate tetrahedral environments. Dynamic-angle-spinning (DAS) NMR provides for high-resolution subspectra of quadrupolar 23Na nuclei allowing spectral separation of multiple distinct sodium environments. Identification of distinct cation sites is important for understanding the physical origin of the `mixed-alkali effect', where non-linear behavior of physical properties is observed in glasses as a function of relative cation populations.

Automated Processing of Two-Dimensional Correlation Spectra

An automated scheme is described which locates the centers of cross peaks in two-dimensional correlation spectra, even under conditions of severe overlap. Double-quantum-filtered correlation (DQ-COSY) spectra have been investigated, but the method is also applicable to TOCSY and NOESY spectra. The search criterion is the intrinsic symmetry (or antisymmetry) of cross-peak multiplets. An initial global search provides the preliminary information to build up a two-dimensional “chemical shift grid.” All genuine cross peaks must be centered at intersections of this grid, a fact that reduces the extent of the subsequent search program enormously. The program recognizes cross peaks by examining the symmetry of signals in a test zone centered at a grid intersection. This “symmetry filter” employs a “lowest value algorithm” to discriminate against overlapping responses from adjacent multiplets. A progressive multiplet subtraction scheme provides further suppression of overlap effects. The processed two-dimensional correlation spectrum represents cross peaks as points at the chemical shift coordinates, with some indication of their relative intensities. Alternatively, the information is presented in the form of a correlation table. The authenticity of a given cross peak is judged by a set of “confidence criteria” expressed as numerical parameters. Experimental results are presented for the 400-MHz double-quantum-filtered COSY spectrum of 4-androsten-3,17-dione, a case where there is severe overlap.

Generalized Two-Dimensional Fourier Transform Infrared Photoacoustic Spectral Depth-Profiling Analysis

This paper reports the first application of a generalized two-dimensional (G2D) correlation method in photoacoustic spectral depth-profiling analysis of laminate/heterogeneous samples. In this method, photoacoustic magnitude spectra at different modulation frequencies are used to generate two-dimensional (2D) correlation spectra. The relative spatial origins of correlated signals are then determined from the signs of the corresponding contours. The unique features and advantages of this technique over previously reported depth-profiling methods are demonstrated and discussed. These include simplification of step-scan photoacoustic phase-modulation experiments as well as 2D correlation-analysis rules, avoidance of phase-related negative-band ambiguities, and enhancement of both spectral and depth resolutions, etc. The recently developed software-based digital signal-processing (DSP) technique for step-scan photoacoustic measurements offers an efficient means (sampling-depth multiplexing advantage) to collect within a single scan all necessary data for this type of 2D correlation analysis. Index Headings: Photoacoustic; PAS; Two-dimensional; Step-scan; FT-IR; Depth profiling; Polymer films; DSP.

New Multinuclear Experiments on Solid Organotin Fluorides

nBu3SnF and Mes3SnF (Mes = mesityl) are organotin fluorides that have direct Sn–F bonds. The119Sn and19F NMR spectra of these compounds have been measured using a selection of CPMAS probes. Doubly decoupled {1H,19F} as well as singly decoupled {1H}119Sn NMR spectra are shown. Double decoupling gives higher resolution spectra and allows the sample of Mes3SnF to be spun slower to give more spinning-sidebands without overlapping of signals, giving improved analysis. The doubly decoupled spectra are sensitive to the power and offset of the decouplers. The effect of off-resonance decoupling has been observed for119Sn NMR through reduced splittings when the19F decoupler power and offset were varied. Proton-decoupled19F spectra have been measured using a new HF double-resonance probe. In addition, two-dimensional heteronuclear correlation spectra are presented for Mes3SnF, allowing a full assignment for the19F and119Sn chemical shifts of the two sites. Full shielding tensor data are reported for both compounds. Values have been derived for the effective (119Sn,19F) dipolar coupling constants from both119Sn and19F experiments, yielding estimates of coupling anisotropy.

Determination of Homonuclear Scalar Coupling Constants by Combining Selective Two-DimensionalNMR Spectroscopy with Convolution and Deconvolution: Applications to Paclitaxel (Taxol)

Homonuclear proton-proton coupling consts. can be detd. accurately by recording selective two-dimensional correlation spectra and by analyzing the resulting multiplets by two-dimensional convolution and deconvolution methods. These methods were evaluated by attempting to det. the complete set of homonuclear couplings in paclitaxel (taxol). Convolution methods have been applied successfully to ten pairs of complementary multiplets recorded by selective correlation spectroscopy (soft-COSY) and pure in-phase correlation spectroscopy (PICSY). These pairs of multiplets gave reliable measurements of ten active couplings. In some cases, satisfactory PICSY spectra could not be obtained, so that the complementary information of soft-COSY and PICSY could not be exploited. Furthermore, couplings involving two strongly-coupled diastereotopic protons (14Ha and 14Hb) could not be detd. in satisfactory manner. Further problems arose due to partial overlap of the chem. shifts of two other protons (10H and 13H). Deconvolution has been applied successfully to all 14 soft-COSY multiplets, yielding 14 ests. of active couplings and 38 ests. of passive splittings. This work provides between one and seven ests. for 21 coupling consts. The accuracy of these different ests. must be weighed with the complexity of the methods used. This work may help to assess the perspectives of completely automated computer-supported anal. of two-dimensional spectra. [on SciFinder (R)]