Projection-reconstruction technique for speeding up multidimensional NMR spectroscopy

The acquisition of multidimensional NMR spectra can be speeded up by a large factor by a projection-reconstruction method related to a technique used in X-ray scanners. The information from a small number of plane projections is used to recreate the full multidimensional spectrum in the familiar format. Projections at any desired angle of incidence are obtained by Fourier transformation of time-domain signals acquired when two or more evolution intervals are incremented simultaneously at different rates. The new technique relies on an established Fourier transform theorem that relates time-domain sections to frequency-domain projections. Recent developments in NMR instrumentation, such as increased resolution and sensitivity, make fast methods for data gathering much more practical for protein and RNA research. Hypercomplex Fourier transformation generates projections in symmetrically related pairs that provide two independent "views" of the spectrum. A new reconstruction algorithm is proposed, based on the inverse Radon transform. Examples are presented of three- and four-dimensional NMR spectra of nuclease A inhibitor reconstructed by this technique with significant savings in measurement time.     

Asymmetric synthesis of trans-3,4-dialkyl-gamma-butyrolactones via an acyl-Claisen and iodolactonization route

[reaction: see text] A new, efficient, and general asymmetric synthesis of enantiomerically pure trans-3,4-dialkyl-gamma-lactones has been developed. The key steps are (1) copper-catalyzed three-component coupling of chiral amine, aldehyde, and alkyne, (2) acyl-Claisen rearrangement, and (3) iodolactonization. The products, chiral gamma-lactones, are versatile synthetic intermediates and structural units of natural products and modified nucleosides.     

Toward amide-modified RNA: synthesis of 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides

Recent discovery of RNA interference has reinvigorated the interest in chemically modified RNA. Chemical approaches may be used to optimize properties of small interfering RNAs, such as thermal stability, cellular delivery, in vivo half-life, and pharmacokinetics. From this perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly interesting modifications that so far have not been tested in RNA interference. Amides are remarkably good mimics of the phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide coupling chemistry. The synthetic challenge that has hampered the progress in this field has been preparation of monomeric building blocks for such couplings, the nucleoside amino acid equivalents. Herein, we report two synthetic routes to enantiomerically pure 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides, monomers for preparation of amide-modified RNA. Modification of uridine, a representative of natural nucleosides, using nitroaldol chemistry gives the target amino acid in 16 steps and 9% overall yield. The alternative synthesis starting from glucose is somewhat less efficient (17 steps and 6% yield of 3'-azidomethyl-5'-carboxy-3',5'-dideoxy uridine), but provides easier access to modified nucleosides having other heterocyclic bases. The syntheses developed herein will allow preparation of amide-modified RNA analogues and exploration of their potential as tools and probes for RNA interference, fundamental biochemistry, and bio- and nanotechnology.     

Frequency-domain Hadamard spectroscopy

A new technique is proposed for multichannel excitation and detection of NMR signals in the frequency domain, an alternative to the widely used pulse-excited Fourier transform method. An extensive array of N radiofrequency irradiation channels covers the spectrum of interest. A selective radiofrequency pulse sequence is applied to each channel, generating a steady-state NMR response acquired one-point-at-a-time in the intervals between pulses. The excitation pattern is repeated N times, phase-encoded according to a Hadamard matrix, and the corresponding N composite responses are decoded by reference to the same matrix. This multiplex technique offers the same sensitivity advantage as conventional Fourier transform spectroscopy. The irradiation pattern may be tailored to concentrate on interesting spectral regions, to facilitate homonuclear double resonance, or to avoid exciting strong solvent peaks. As no free induction decay is involved, the new method avoids problems of pulse breakthrough or lineshape distortion by premature termination of the time-domain signal.     

Two-dimensional Hadamard spectroscopy

Direct frequency-domain excitation of NMR with an array of different radiofrequencies has been used to speed up two-dimensional NMR experiments by a large factor. Multiplex excitation in the F(1) frequency dimension is restricted to the signal-bearing regions and is encoded according to a Hadamard matrix of dimension N by N, where N is a relatively small number. The detected signals are decoded by reference to the same Hadamard matrix. Alternatively a phase-encoding scheme can be employed. Two-dimensional correlation experiments (COSY and TOCSY) and cross-relaxation measurements (NOESY) implemented on proton systems can be completed in less than a minute in cases where the intrinsic sensitivity is sufficiently high that prolonged multiscan averaging is not required. The results are presented in the form of a high-resolution contour diagram similar to the familiar two-dimensional spectra obtained by Fourier transform methods. Experiments on strychnine demonstrate more than two orders of magnitude improvement in speed compared with the traditional methods.     

Resolving ambiguities in two-dimensional NMR spectra: the 'TILT' experiment

Ambiguities in two-dimensional nuclear magnetic resonance spectra due to overlap are usually resolved by recording a three-dimensional version of the experiment. It is shown that a simpler solution is to record a tilted projection of the three-dimensional spectrum, derived by Fourier transformation of the time-domain signal acquired while the two evolution parameters are varied simultaneously at the appropriate rates. By avoiding the need to record the full three-dimensional spectrum, this saves an order of magnitude in measurement time. The tilt technique is illustrated by reference to degenerate responses in the TOCSY and NOESY spectra of a small protein, agitoxin, where the 1H and 15N frequencies are incremented in tandem.     

Exploiting natural abundance 13C–15N coupling as a method for identification of nitrogen heterocycles: practical use of the HCNMBC sequence

In this paper, we detail the results of ¹H–¹⁵N correlation data obtained via ¹³C–¹⁵N coupling at natural abundance on a number of classes of azoles including pyrazoles, imidazoles and triazoles. The experiment produces data that is highly complementary to direct ¹H–¹⁵N HMBC type correlations in that it can provide ¹⁵N chemical shift data for nitrogen that may not show up in the HMBC. This is particularly advantageous in the triazoles where ¹⁵N chemical shift can be diagnostic of regiochemistry. Because of the consistency in J_CN values among the azoles, the experiment produces distinctive correlation patterns that can be used for identification of regiochemistry. The experiment can also be used to directly measure ¹³C–¹⁵N coupling constants. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultrafast-based projection-reconstruction three-dimensional nuclear magnetic resonance spectroscopy

Recent years have witnessed increased efforts toward the accelerated acquisition of multidimensional nuclear magnetic resonance (nD NMR) spectra. Among the methods proposed to speed up these NMR experiments is "projection reconstruction," a scheme based on the acquisition of a reduced number of two-dimensional (2D) NMR data sets constituting cross sections of the nD time domain being sought. Another proposition involves "ultrafast" spectroscopy, capable of completing nD NMR acquisitions within a single scan. Potential limitations of these approaches include the need for a relatively slow 2D-type serial data collection procedure in the former case, and a need for at least n high-performance, linearly independent gradients and a sufficiently high sensitivity in the latter. The present study introduces a new scheme that comes to address these limitations, by combining the basic features of the projection reconstruction and the ultrafast approaches into a single, unified nD NMR experiment. In the resulting method each member within the series of 2D cross sections required by projection reconstruction to deliver the nD NMR spectrum being sought, is acquired within a single scan with the aid of the 2D ultrafast protocol. Full nD NMR spectra can thus become available by backprojecting a small number of 2D sets, collected using a minimum number of scans. Principles, opportunities, and limitations of the resulting approach, together with demonstrations of its practical advantages, are here discussed and illustrated with a series of three-dimensional homo- and heteronuclear NMR correlation experiments.