Phosphorus-31, heteronuclear coupling

As we have already pointed out in the section dealing with heteronuclear coupling that it is not always necessary to confirm the presence of a particular hetero atom by acquiring the NMR spectrum of that nucleus. More often than not, the hetero atom will have a clear signature in the proton or carbon spectrum. Fluorine and phosphorus are both examples of nuclei that couple to protons over two, three, four and even more bonds. 

The use of phosphonodifluorodithioacetate as a 2ji component in cycloadditions with a variety of dienes provides simultaneous direct access to fluorine and phosphorus containing 3,6-dihydro-2/7-thiopyrans, for example, 217 and their 3-ones 218. Complex signals result from the heteronuclear couplings <2002TL2033>. 

Heteronuclear coupling of to and to was discussed in Chapter 6, Sections 6.15 and 6.16. Fluorine-19 is the only natural isotope for this element, and while phosphorus has more than one isotope, phosphorus-31 is the most abundant. This section will cover couplings to F and Since F and P each have a nuclear spin of 1/2, the principles discussed earlier in this chapter apply directly to interpreting resonances coupled to these elements. 

Both types of experiments detect magnetization arising from mutually coupled heteronuclear spin-triples. Even if the spectra can in principle be displayed in a 3D-cube representation (Rg. 10), the analysis of 2D slices or projections is often preferred for the interpretation. Important features can be derived as is demonstrated in the example shown in Fig. 10, e.g. from inspection of H,C planes taken at the chemical shift of an individual phosphorus atom, which give a two-dimensional H,C correlation of all signals... 

It has been shown by Trebosc et that the FS REDOR (Frequency Selective Rotational Echo Double Resonance) experiment can be used for accurate through-space measurements in spin pairs that involve the quadrupolar nuclei. The experiment reveals both heteronuclear dipolar and scalar couplings, which can be re-introduced selectively site after site. As an example, couplings between the aluminium and phosphorus nuclei in the VPI5 zeohte have been measured. They agree very well with those reported in the literature, which validates the authors approach. 

Heteronuclear NMR spectroscopy - Al and P MAS NMR correlation spectroscopy - in which dipolar (CP, TEDOR experiments) or J-coupling (the INEPT experiment) is used to determine the correlation between different A1 and P sites,can assist in the NMR crystallography of aluminophos-phates. For AIPO4-I4, for example, Wiench and Pruski describe a series of NMR experiments that use polarisation transfer from the quadrupolar Al to spin- P via J-coupling to confirm the connectivity between aluminium and phosphorus sites in the structure. 

Bolton et al. (1982) have also performed P-NMR experiments with messenger ribonucleoprotein (mRNP), which is about the same size as ribosomes. The T, NOE, T, R, and lP,/2 values are Usted in Table IV. The P r, value of mRNP is much smaller than that of any other nucleic acid-protein complex in Table IV in fact, it is smaller than one could expect for a P nucleus relaxed solely by protons on the RNA. Consequently, selective P( H NOE experiments were carried out in which the 3 P resonance intensity was monitored, as 50-Hz windows in the H-NMR spectrum were strongly irradiated rather than irradiating all proton frequencies, as is usually done with heteronuclear NOE experiments. The selective NOE experiments showed that the phosphorus in the mRNP was dipolar-coupled to protein protons as well as ribose protons. Apparently, the nature of the protein-nucleic add interactions differ in comparison with the other nucleic add-protdn complexes of Table IV, in that protons from the protein are much closer to the phosphorus of RNA. 

Fig. 14. (Left) Heteronuclear, correlated absolute-value spectrum of guanosine 2 -monophosphate. The p2 dimension contains the H-coupled P spectrum and the F, dimension contains the P-coupled H spectrum. The large peaks at 4.6 ppm (F,) are unsuppressed axial peaks. (Right) Phase-sensitive slices for each phosphorus transition b and c in the 2 -GMP two-dimensional spectrum. Spectra a and d are simulated for comparison with the experimental slices b and c. From Bolton and Bodenhausen (1979). Copyright 1979 American Chemical Society.

Because the P and H chemical shifts are correlated, additional data are available for the assignment of monophosphorylated cell metabolites. The Q -3ip resonances of many molecules with phosphate chains will also appear in the heteronuclear chemical-shift correlation map. Cross-sections of the P dimension contain H- H coupled spectra. Obtaining H-NMR parameters from P cross-sections in the two-dimensional data set has several advantages for biological systems. Only protons that are spin-spin coupled to a phosphorus appear in the two-dimensional slices. Ribose protons in nucleotides, oligonucleotides, and other phosphorylated molecules resonate... 

Phosphoserine provides a stringent test of the two-dimensional technique (Bolton, 1981a). Not only is the phosphorus resonance in phosphoserine coupled to three nonequivalent protons, but the two methylene protons are also strongly coupled to each other. Figure 16 contains simulated P spectra using the parameters in Table I. The agreement with the experimental two-dimensional difference spectra in Fig. 17 is impressive. The application of heteronuclear two-dimensional spectroscopy permits one to obtain accurate proton coupling constants that may be used to evaluate the conformation of phosphorylated cellular metabolites. 

Fig. 18. Heteronuclear two imensional relayed coherence-transfer spectrum of phos-phothreonine. The absolute-value contour plot (bottom) contains the phosphorus doublet on the vertical axis and the proton spectrum on the horizontal axis. The scalar coupling patterns for protons indirectly coupled to the phosphate group can be evaluated from the cross-section (top). From Bolton and Bodenhausen (1982b).

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