5.8: NMR de moléculas fosforiladas

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Because so many biological molecules contain phosphoryl groups, it is worthwhile to look at how scientists use NMR to determine the structure of these molecules. Consider the case of isopentenyl diphosphate, the building block molecule used by cells to make 'isoprenoid' compounds such as cholesterol (in many animals), or beta-carotene (in some plants). NMR spectra of this molecule were taken in a D₂O solvent, buffered with ND₄OD (the deuterium equivalent of aqueous ammonium hydroxide, NH₄OH) (J. Org. Chem. 1986, 51, 4768).

¹H-NMR

H_a: 4.05 ppm (td); ³J_Ha-Hb = 6.6 Hz; ³J_Ha-P_A= 3.3 Hz.

H_b: 2.39 ppm (t) ³J_Ha-Hb = 6.6 Hz

H_c: 4.86 ppm (s)

H_d: 1.77 ppm (s)

The ¹H-NMR signals for H_b, H_c, and H_d are what we would expect given our present knowledge. Why, though, is the signal for H_a split into a triplet of doublets (td)? First of all, as, expected, the two neighboring H_b protons split the H_a signal into a triplet, with ³J = 6.6 Hz. Then, the signal is further split into doublets (³J = 3.3 Hz) by P_A, the closer of the two phosphorus atoms.

Recall that the most abundant isotope of phosphorus is ³¹P, which is NMR active - it has a magnetic dipole just like ¹³C and ¹H, and thus will participate in spin-spin coupling interactions with nearby protons and ¹³C nuclei.

In the ¹³C spectrum, notice that the signals for both C₁ and C₂ are split into doublets by the magnetic field of P_A, through 2-bond and 3-bond coupling, respectively.

¹³C-NMR (proton-decoupled)

C₁: 40.7 ppm (d); ²J_C₁_-P_A= 7.2 Hz

C₂: 67.0 ppm (d); ³J_C₂_-P_A= 4.0 Hz

C₃: 147.4 ppm

C₄: 114.6 ppm

C₅: 24.5 ppm

This splitting is observed even in the proton-decoupled spectrum - carbon-phosphorus coupling is not turned off.

Because ³¹P is NMR-active, we can also, with the right instrumentation, directly observe the phosphorus NMR signals. In an NMR instrument with a 7.1 Tesla magnet, where protons resonate at 300 MHz and ¹³C nuclei resonate at 75 MHz, phosphorus resonates at 32 MHz. In ³¹P-NMR experiments, the reference standard used to determine the 0 ppm point is usually phosphoric acid (TMS, the standard for ¹H- and ¹³C-NMR, doesn't have a phosphorus atom!). The ³¹P-NMR spectrum of isopentenyl diphosphate has, as expected, two peaks, each of which is upfield of the phosphoric acid standard (negative chemical shifts!) and split into a doublet (²J_P-P = 20 Hz) due to coupling between the two phosphorus nuclei.

P_A: -11.03 ppm (d)

P_B: -7.23 ppm (d)

²J _P_A_-P_B= 20 Hz

Notice that although the C₁ and C₂ signals were split by P_A in our ¹³C-NMR spectrum, in the ³¹P-NMR spectrum the P_A signal is not split by the nearby carbons - this is because, of course, both of these carbons are the NMR-inactive ¹²C isotope in 99 out of 100 molecules. In addition, P-H splitting is not observed in this ³¹P spectrum, because proton decoupling is in effect.

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