J tuning of SUPERCOSY as an aid to resonance assignments

Abstract

Nuclear magnetic resonance spectroscopy has been extensively used for conformational analysis of biological molecules (I). With the advent of two-dimensional NMR techniques, it has become possible to obtain individual resonance assignments and detailed structural information on large molecules having molecular weights of the order of 6000-7000 (2-15). Recently a pulse sequence has been proposed (26) which is superior to the normal COSY (I 7, 18) scheme for observation of coupling correlations in complicated spin systems. The new pulse scheme has the sequence: 90-t,-A-180-A-90-A-180-At2, where A is a fixed delay and t, and t2, are, respectively, the usual evolution and detection periods of two-dimensional spectroscopy. The value of A depends upon the nature of the spin systems and the magnitudes of the coupling constants (J) between the spins. For an AX spin system, the optimum value of A is 1/4J and for an AX2 system, it is 1/2J. The main advantage of SUPERCOSY over the COSY scheme lies in the fact that the cross-peaks and the diagonal peaks in SUPERCOSY consist of in-phase and antiphase components respectively, while exactly the opposite is true in the COSY spectrum. Consequently, under conditions of overlap or poor digital resolution, cross-peaks build much faster in intensity, while the diagonal peaks tend to grow relatively slower. The fact that the fixed delay A in the SUPERCOSY pulse scheme depends on the value of J enables one to tune the experiment to a particular value of J which may be of special interest. In other words, cross-peaks arising from particular values of J can be selectively enhanced. We have used this principle here to observe selective proton couplings so as to arrive at a unique resonance assignment of the ring protons in the tryptophan residue of a decapeptide, leutinizing hormone releasing hormone (LHRH). Figure 1 shows SUPERCOSY spectra for two different values of A, 30 ms (A) and 40 ms (B). While the complete analysis will be published separately, the intention of this note is to demonstrate how J tuning of SUPERCOSY can be used as an aid in resonance assignment procedures. The two spectra shown in Fig. 1 depict variations in the intensities of several cross-peaks and diagonal peaks. Figure 2 shows expansions of the aromatic regions of the spectra (02 = 6.4-l 1 .O ppm; WI = 6.4-l 1 .O ppm). The peaks labeled A, B, C, D, etc, identify the J-coupling correlations between various ring protons. For example, in the spectrum of Fig. 2A,