Although organic chemists typically focus on hydrogen and carbon when
analyzing complex molecules, there is an increasing amount of analysis
done on other elements such as phosphorous or sulfur. NMR is an
underappreciated tool to understand the role of these heteroatoms,
especially phosphorous, whose NMR-active isotopes are abundant and have
high gyromagnetic ratios.
Adenosine triphosphate (ATP) is ubiquitous in eukaryotic and
prokaryotic cells and serves as an energy source. Its three phosphate
groups and purine ring serve as a great template for some interesting
NMR experiments and demonstration of how heteronuclear and heteronuclear
2-dimensional experiments can be used to elucidate molecular structure.
Figure 1:
1H NMR spectrum of 10 mg/ml ATP in D
2O.
The
1H NMR spectrum of ATP is straightforward to acquire
as long as one doesn’t mind losing the exchangable protons to the
solvent. Water suppression techniques are unnecessary given the
relatively sharp solvent signal (more on water suppression in a future
post). The small peaks at δ 3.5 and d 1.2 ppm are residual triethylamine
from the commercial material. Assignment of the non-aromatic peaks
(H-1′, H-2′, H-3′, H-4′, H-5′) can be achieved using COSY but will not
be shown here – see
previous post on clarithromycin for details on
1H assignment using COSY.
Figure 2:
13C NMR spectrum, with the doublet at δ 84 ppm shown in inset
Likewise, the
13C NMR of ATP is straightforward to acquire
with an overnight scan. A small amount of line broadening (LB = 1.0 Hz)
was applied to improve the signal-to-noise, but limited to keep the
maintain resolution of the doublet at δ 84 ppm (which arises due to
13C-
31P coupling).
Figure 3:
1H-
13C HSQC spectrum
The alkyl
13C resonances can easily be assigned using an HSQC spectrum, which correlates the
1H spectrum with the
13C spectrum. In this phase-sensitive version of the HSQC experiment, red cross-peaks have CH
2 multiplicity (H-5′ / C-5′) while black cross-peaks have CH or CH
3 multiplicity. The
13C
resonances at δ 142 and d 145 ppm are C-2 and C-8 (but assignment not
yet known), while the resonances at δ 118, 148, and 150 ppm do not have
protons bonded (C-4, C-5, and C-6; assignment not yet known).
Figure 4:
1H-
13C HMBC spectrum
Heteronuclear
Multiple
Bond
Correlation
is similar to the HSQC, except that 1-bond correlations are canceled
out, and only 2- and 3-bond correlations are shown. The HMBC experiment
allows one to identify all of the aromatic resonances, on both the
1H and
13C
spectra. The H-1′ (δ 6.0 ppm) signal show correlations to the C-4 (δ
148 ppm) and C-8 (δ 142 ppm) signals. The H-8 (δ 8.5 ppm) signal shows
correlations to the C-4 and C-5 (δ 118 ppm) signals. The H-2 (δ 8.3 ppm)
signal shows correlations to the C-4 and C-6 (δ 150 ppm) signals, as
well as a weak signal to C-5.
Now that the
1H and
13C spectra are fully assigned, we can direct our attention to the
31P NMR spectra.
Figure 5:
31P NMR spectrum
31P has decent sensitivity and high abundance and is a
good nucleus to use as an example of heteronuclear spectroscopy. A
1-dimensional
31P spectrum shows three signals at δ -10, -11, and -23 ppm. This spectrum is
1H-coupled, but the 3-bond
1H-
31P coupling is too small to see. The observed couplings are
31P-
31P
couplings, and based on the multiplicity, it is clear that the middle
phosphate gives rise to the signal at δ -23 ppm. The other two signals
can be differentiated using HMBC, except this time
1H-
31P instead of
1H-
13C.
As an internal standard, approximately 0.5 equivalents of trimethylphosphonoacetate were added. The
31P
signal at δ +26.06 ppm gives strong crosspeaks to the methylene (δ 3.2
ppm) as well as methyl (δ 3.8 ppm) protons and helps calibrate the
spectrum along the
31P axis.
Figure 6:
1H-
31P HMBC spectrum, full (top) and enlarged on the ATP signals (bottom).
In this case, crosspeaks between the
31P signal at δ -11.27 ppm and the H-4′ and H-5′ signals from δ 4.15-4.35 ppm were observed. The crosspeaks establish the
31P signal at δ -11.27 ppm as the first phosphate, allowing assignment of the
31P NMR spectrum as well as confirmation that the triphosphate group is covalently attached to the nucleoside via that 5′-OH.