Two-dimensional NMR spectroscopy with soft pulses

Abstract

Two-dimensional spectroscopy is today routinely used in order to facilitate the assignment of resonance lines in NMR spectra and to determine molecular structure (1, 2). Homonuclear correlation spectroscopy (COSY) and NOE spectroscopy (NOESY), for example, are the two pivotal methods in the structural analysis of biological macromolecules in solution (3). Whenever a detailed analysis of the cross-peak multiplet structure is desired, e.g., for the quantitative determination of coupling constants, high-resolution 2D spectra are required. High resolution may also favor sensitivity in cases where the cross peaks are antiphase as in COSY spectra. High-resolution 2D spectroscopy is, however, often faced with practical limitations of data storage and excessive measurement time caused by the large number of required t, experiments. We propose in this communication to employ frequency-selective soft radiofrequency pulses in order to circumvent the mentioned limitations. When excitation and detection are focused to a small area of the 2D spectrum, high resolution can be achieved by concentrating all available data points to this area. O ther selective experiments derived from 2D spectroscopy have recently been proposed by Kessler et al. (4). These experiments result, however, in one-dimensional spectra corresponding to cross sections through 2D spectra. The experiment described in this communication, on the other hand, delivers true 2D spectra of restricted frequency ranges. Two types of frequency selectivity in 2D spectra are illustrated in Fig. 1. In Fig. 1 a a restricted frequency range A& is excited in o1 by selective preparation pulses resulting in a band parallel to the w2 axis. If it is desirable to restrict also the frequency range in w2, selective pulses can be. incorporated into the mixing sequence in order to allow coherence transfer only between the ranges AQ, and Aa2 (Fig. lb). Alternatively, w2 selectivity can be achieved by means of audiofrequency filters; however, in some cases it is achieved at the expense of sensitivity as will become clear in the following. Frequency selection with soft pulses throughout the entire pulse sequence offers in addition the possibility to interleave experiments for selection of different nonoverlapping spectral regions thus avoiding relaxation delays between experiments. It is evident that the principles above are applicable to all types of 2D experiments.