Two-bond Correlations Research Articles

Clean PDI-1 SQ: Suppression of HSQC artifacts in 2D proton-detected INADEQUATE spectra by pulse sequence redesign

Proton-detected INADEQUATE NMR experiments are widely used for structure elucidation of small molecules, in particular the implementations that display 13C single-quantum rather than double-quantum frequencies in the indirect dimension of 2D spectra. But unfortunately, such spectra in addition to the desired 1H–13C two-bond correlations also contain HSQC artifacts of comparable magnitude. The redesigned versatile experiment presented in this paper requires no compromise based on different 13C multiplicities and suppresses the HSQC artifacts that are a source of possible spectral misinterpretation. Demonstration of the new method is shown by applications to typical small molecules of different complexity.

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC

HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations. There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed.

Comparison of the Effects of Different 19F π Pulses on the Sensitivity and Phaseability of the 19F-13C HSQC

The 19F-13C heteronuclear single quantum coherence (HSQC) experiment is vital for the structural elucidation of polyfluorinated organic species, yet its sensitivity and phaseability are limited by difficulties in uniform excitation of the widely disperse 19F spectral window. Adiabatic pulses of different types have previously been employed to generate effective π pulses for inversion and refocussing, but a systematic comparison of various adiabatic and other inversion pulses has not been published. In this work, it was observed that the use of a broadband inversion pulse (BIP) during the t 1 evolution period resulted in properly phaseable spectra for experiments optimized to detect 1 J CF, in contrast to CHIRP or WURST adiabatic pulses. For the INEPT and reverse-INEPT transfer segments of the HSQC, optimal sensitivity for resonances distant from the transmitter frequency was afforded by optimized universal rotation (BURBOP) or CHIRP pulses. In HSQC experiments with delays optimized for two-bond correlations, only the use of BURBOP pulses in INEPT and reverse-INEPT sequences afforded spectra cleanly phaseable across the F 2 and F 1 spectral windows. This observation is supported by off-resonance pulsed field gradient spin-echo experiments.

Symmetry-based dipolar recoupling by optimal control: Band-selective experiments for assignment of solid-state NMR spectra of proteins

We present design of novel low-power homonuclear dipolar recoupling experiments for magic-angle-spinning solid-state NMR studies of proteins. The pulse sequences are developed by combining principles of symmetry-based dipolar recoupling and optimal control-based pulse sequence design. The scaffold of the pulse sequences is formed by known CN-type recoupling sequences, while the intrinsic sequence elements are designed using optimal control. This procedure allows for the development of high-performance pulse sequences demanding significantly weaker rf fields than previous symmetry-based pulse sequences while compensating for rf inhomogeneity and providing excitation over relevant ranges of chemical shifts for biological applications. The new recoupling experiments, referred to as optimal control CN ((OC)CN), are demonstrated numerically and experimentally by two-dimensional (2D) (13)C-(13)C and three-dimensional (3D) (15)N-(13)C-(13)C chemical shift correlation experiments on uniformly (13)C, (15)N-labeled ubiquitin. Exploiting the double-quantum, band-selective dipolar recoupling properties of the (OC)CN experiments, we demonstrate significant sensitivity enhancement for 2D and 3D correlation spectra showing exclusively one- or two-bond correlations.

HAT HMBC: A hybrid of H2BC and HMBC overcoming shortcomings of both

A new 2D NMR experiment, HAT HMBC, that is a hybrid of H2BC and HMBC aims at establishing two-bond correlations absent in H2BC spectra because of vanishing 3 J HH coupling constants. The basic idea is to create an additional π phase difference in the multiplet structure in HMBC peaks with respect to the n+1 J HH coupling constant between the proton(s) attached to a 13C and a 1H separated by n bonds. Thus HMBC peaks associated with small J HH will be the most attenuated in a HAT HMBC spectrum in comparison to a regular HMBC spectrum, i.e. peaks associated with n+1 J HH and n J CH will for n > 2 usually be strongly attenuated. The HAT HMBC pulse sequences contain the same number of pulses as regular HMBC and are only a few milliseconds longer.

H2BC: a new technique for NMR analysis of complex carbohydrates

It is demonstrated that the H2BC NMR pulse sequence ( J. Am. Chem. Soc. 2005, 127, 6154, Magn. Reson. Chem. 2005, 43, 971–974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing 2 J CH coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC–TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC–TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.

Heteronuclear Two-Bond Correlation: Suppressing Heteronuclear Three-Bond or Higher NMR Correlations while Enhancing Two-Bond Correlations Even for Vanishing 2JCH

A novel two-dimensional NMR pulse sequence, H2BC, for long-range correlation of 1H and 13C nuclei is presented. The experiment has several attractive features compared to the widely used HMBC experiment, for example, (a) typically strong enhancement of correlations over two bonds while suppressing those over more bonds, that is, resolving ambiguities in standard HMBC spectra and showing two-bond correlations not present in HMBC spectra, (b) independence of long-range 1H-13C coupling constants, (c) full homo- and heteronuclear decoupling in the indirect dimension and heteronuclear decoupling in the acquisition dimension, (d) pure 2D absorption peak shapes, and (e) a pulse sequence duration significantly shorter than that of HMBC. The experiment is quite complementary to HMBC and does not effect correlations to quaternary carbons that must be obtained by HMBC.

13C-13C NOESY: A constructive use of 13C-13C spin-diffusion

(13)C-(13)C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, (15)N, (13C) and 70% (2)H labeled). (13)C-(13)C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time tau(m) of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C' and C(beta). The interpretation was confirmed by simulations. This approach broadens the range of applicability of (13)C-(13)C NOESY spectroscopy.

Differentiation of HMBC two- and three-bond correlations: A method to simplify the structure determination of natural products

The structure elucidation of natural products, today, relies heavily on the application of proton-detected heteronuclear NMR experiments. Perhaps the most useful of these methods is the HMBC experiment, which provides correlations between protons and carbons over two and three bonds. The application of the HMBC method for the direct translation of H,C correlations to yield bonding information is limited, however, by the fact that it does not distinguish between 2 J CH and 3 J CH correlations. Reported here is an application of the recently described 1,1-ADEQUATE experiment that yields only two bond H,C connectivities in HCC moieties and therefore allows the differentiation of HMBC two- and three-bond correlations. The method is demonstrated on a 14 mg sample of a new marine natural product, 5,6-dihydro lamellarin H ( 1). An NMR methodology is presented which allows the differentiation of two- and three-bond correlation in HMBC spectra. The ω 1-refocused 1,1-ADEQUATE is used to obtain exclusively two-bond correlations which thereby facilitates the assignment of the constitution of complex structures. The method is demonstrated on a new polycyclic marine natural product, 5,6-dihydro lamellarin H.

J-Compensated Proton-Detected Heteronuclear Shift-Correlation Experiments

A new proton-detected heteronuclear shift-correlation experiment, J-HSMQC, is proposed for sensitivity enhancement for samples containing a wide range of 1 J CH values. This method, which incorporates only two additional τ periods and has the same number of RF pulses as the HSQC experiment, is found to give significant enhancements for CH fragments with 1 J CH values which deviate considerably from the coupling constant for which τ is optimal. The J-HSMQC maps also contain reasonably intense two-bond correlations for some CH fragments.