Published on Web 09/01/2006
DFTMP, an NMR Reagent for Assessing the Near-Neutral pH of Biological
Samples
Michael D. Reily,* Lora C. Robosky, Matthew L. Manning, Andrew Butler,† John David Baker, and
R. Thomas Winters
Pfizer Global Research and DeVelopment, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received May 30, 2006; E-mail: michael.reily@pfizer.com
Scheme 1
NMR has played a central role in our understanding of biological
systems. In particular, high-resolution NMR is quite useful at
elucidating atomic-level dynamic information and quantitative
concentration information on complex systems such as drug-target
interactions, enzymatic and nonenzymatic kinetics, or biological
fluids analysis. One important factor in such biochemical systems
is sample pH, which affects both biological function and specific
NMR parameters. Slight perturbations in the electron distribution
around an NMR-active atomic nucleus result in measurable changes
in its resonant frequency. Hence it is possible to evaluate events
such as ionization and protonation state through changes in the
chemical shift (∆δ) of nonexchangeable nuclei proximal to the locus
of the perturbation. The power of NMR to probe site specific acid-
base properties was recognized early, and advancements over the
pH and divalent metal ion content. An accurate knowledge of the
urinary pH can both identify abnormal conditions and provide a
context upon which to make peak assignments for species whose
last few decades have positioned NMR as the most powerful tool
chemical shifts are pH sensitive.
to carry out such investigations.1
To evaluate the utility of 1 for in situ pH measurements using
In practice, pH measurements are typically made using conven-
tional pH electrodes or by the addition of internal standards that
have chemical shifts which are sensitive to pH in and around the
pH of interest (such as imidazole for near neutral pH2). The former
approach has the advantage of direct pH measurement with a tool
of determinable accuracy but can be tedious and requires sample
manipulation and direct contact with the electrode which can result
in sample loss and contamination. The latter avoids these risks but
1
NMR, H NMR spectra were recorded on a human urine sample
5
containing 1 and DSS-d6 over a range of pH. Individual aliquots
of a single urine sample were mixed with measured amounts of
HCl or NaOH then diluted with distilled water. A 550 µL aliquot
of the pH-adjusted urine sample was mixed with 65 µL of 3.0 mM
DSS-d6 in D2O and 35 µL of 1.8 mM 1 in H2O. Proton spectra
were immediately recorded on a Bruker AV-600 NMR spectrometer
at 19.0 °C. The pH of each sample was measured by a Tecan robotic
system at 19.0 °C immediately after each NMR spectrum was
acquired using a WTW inoLab pH level 2 m in conjunction with
can introduce extraneous signals that may overlap analyte signals
of interest in the NMR spectrum.
This situation has stimulated us to seek a novel chemical shift-
a Schott N 5900 A-2m (Ag/AgCl) electrode that was calibrated
sensitive reagent for in situ pH measurement in NMR experiments
prior to the first sample measurement. The pH of the calibration
conducted at near-neutral pH. Such a compound must be highly
buffers was remeasured after the last sample and found to be within
stable and have chemical shifts that (1) do not overlap with analyte
the acceptable range.
components, (2) are sensitive to pH (>0.005 ppm/pH unit) in the
The chemical shift versus pH data for 1 relative to DSS can be
adequately modeled using a simple 3-term thermodynamic relation-
pH 6-8 range, and (3) are insensitive to Ca2+ and Mg2+ concentra-
tions often found in biological samples. We have synthesized and
ship (eq 1), where pKa is the pKa of first deprotonation of 1, dobs
,
characterized a new chemical entity that meets all of these require-
ments, 1,1-difluoro-1-trimethylsilanyl methylphosphonic acid (DFT-
MP) 1, (Scheme 1). The nine equivalent protons of the trimethylsilyl
group offers a single strong, sharp resonance at ∼0.2 ppm. An
additional characteristic of this compound is that it contains proton,
fluorine, and phosphorus nuclei and can thus serve as a chemical
shift reference point and pH indicator for multinuclear studies.
One NMR application that has recently garnered a great deal of
interest is the quantitative analysis of endogenous metabolites in
biological fluids and the subsequent correlation of changes in
biomolecular composition with physiological or genetic perturba-
tion.3 A common problem with this approach is the pH and metal
ion dependence of the chemical shift of individual components,4
which confounds the identification of endogenous analytes in
complex matrixes that often contain hundreds of molecules. This
is especially true in urine, which can have rather wide ranges in
d
min, dmax are the observed, fully protonated and fully (mono)
deprotonated chemical shift of 1, respectively.
δmin - δobs
δobs - δmax
0.224 - δ
δ - 0.193
pH ) pKa + log
) 6.246 + log
(1)
[
]
[
]
The experimental data and predictive model are shown in Figure
1. The chemical shift of the single resonance from the nine
equivalent trimethylsilyl protons ranges from 0.195 and 0.225 ppm,
with excellent agreement between calculated and observed pH
between pH 4.3 and 8.2. Outside these ranges, the ∆δ with pH
becomes small and the model loses accuracy. Between pH 5 and 8
the RMSE error of pH prediction is 0.02 pH units, which is
comparable to the accepted accuracy of conventional glass elec-
trodes.1 The overall titration shift between extremes (pH ∼4 to 9)
is ∼0.03 ppm with a maximum slope of ∼0.015 ppm/pH unit
between pH 5.7 and 7.4 and is sufficiently large for accurate
† Current Address: MS 8274-1341 Eastern Point Road, Groton, CT 06340.
9
12360
J. AM. CHEM. SOC. 2006, 128, 12360-12361
10.1021/ja063773h CCC: $33.50 © 2006 American Chemical Society
Reily, Michael D.
