Multiplicity proton resonance assignment

The practical use and the advantage of proton off-resonance decoupling - less multiplet overcrowding and more signal noise relative to coupled spectra - is illustrated in Fig. 2.47. for a triterpene derivative in comparison to modern and more accurate methods for determination of CH multiplicity. An unequivocal assignment of the number of directly attached hydrogens may be possible for all carbons. 

Nuclear coupling between 13C nuclei and directly bonded protons (a relatively strong interaction) causes the multiplicity of lines - sometimes a helpful effect in making resonance assignments. 

Figure 13(b) shows a JH—15N HSQC spectrum acquired from 0.5 mmol l-1 sample of a 41-residue peptide toxin from the spider Agelena orientalis. The toxin was produced recombinantly and uniformly labeled with 15N. This HSQC spectrum was collected in 30 min, compared with the 12 h required to acquire a natural abundance spectrum from an unlabeled sample of equivalent concentration (see Figure 11). The HSQC, together with the related heteronuclear multiple quantum coherence (HMQC)54 experiment, forms the cornerstone of a wide range of 2D, 3D, and 4D experiments that are designed to facilitate sequence-specific resonance assignment and determination of protein structure. Note that the HSQC technique is the technique of choice for correlation of H and 15N shifts due to generally narrower linewidths in the 15N dimension.55,56 Furthermore, because these and most of the other heteronuclear experiments described below are designed to observe amide protons, the sample must be in H20 (rather than D20). Consequently, a means of suppressing the H20 resonance is required (for details see Section 9.09.2.6).

The C line assignments were made from the combination of DEPT and 2D C- H correlated spectroscopy despite the complexity of the conventional C spectrum. DEPT spectroscopy allowed the multiplicity of each resonance to be determined unambiguously. Hence, C assignments were made easily from the 2D C- H correlated spectrum even in situations where overlap of methine and methylene signals occurs in the proton spectrum. Furthermore, equivalent and nonequivalent methylenes were distinguished in the 2D C- H correlated spectrum, and this allowed assignments to be made despite spectral overlap of proton resonances. Proton chemical shifts were determined more accurately from the correlated... 

The T resonances in the 73-74 ppm region have multiple-bond correlations in the HSQC-TOCSY spectmm to proton resonances of S and/or S , methylenes. Therefore, they are attributed to Xm groups such as those found in stmcture 20. Analysis of these data provided resonance assignments for all the S and T type carbons for the stmctures in Scheme 1. Detailed analysis of expansions of the peak-containing regions of the 2D-NMR data provided complete resonance assignments for all monomer sequences up to the tetrad level in poly(EV). 

Proton magnetic resonance (chloroform-d) S (multiplicity, number of protons, assignment) 3.30 (singlet, 12, OC/fg), 6.10 (singlet, 4, ring protons.)... 

The mother liquor and wash solution are combined and concentrated to 200 ml. on a rotary evaporator. Upon cooling, a second crop (15-18 g.) of product is obtained. This second crop was a semisolid material. The spectral properties of the crystalline product are as follows infrared (Nujol) cm.-1 1660 (0=0) proton magnetic resonance (chloroform-d) <5, multiplicity, assignment 1.45 [singlet, C (C f/3)3],... 

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