Fluorine NMR reporter for phosphate anions

Article abstract of DOI:10.1039/c3cc42169d

A fluorine-labelled zinc(ii)-dipicolylamine coordination complex reports the presence of phosphate anions in aqueous solution, especially pyrophosphate and ADP, and is used to monitor the enzymatic hydrolysis of ATP. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc42169d

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Fluorine NMR reporter for phosphate anions†
Haiying Gan, Allen G. Oliver and Bradley D. Smith*
Cite this: Chem. Commun., 2013,
49, 5070
Received 25th March 2013,
Accepted 19th April 2013
DOI: 10.1039/c3cc42169d
www.rsc.org/chemcomm
A fluorine-labelled zinc(II)-dipicolylamine coordination complex
reports the presence of phosphate anions in aqueous solution,
especially pyrophosphate and ADP, and is used to monitor the
enzymatic hydrolysis of ATP.
19F NMR has many attractive features that encourage its use in
biomedicine.1 The 19F nucleus has a natural abundance of
Fig. 1 Reversible association of 19F NMR reporter 1 with a bidentate anion.
1
100% and signal sensitivity of 83% relative to H NMR. 19F NMR
spectra of small molecule samples at submillimolar concentrations The few literature examples of this strategy include 19F labelled
can be acquired with high signal to noise. Furthermore, recent reporters that respond reversibly to temperature,7 pH,8 metal
breakthroughs in NMR signal enhancement, using techniques that cations,9 diol-containing molecules,10 and protease enzymes.11
exploit dynamic nuclear polarization, allow detection of 19F signals
Herein, we describe the recognition properties of compound
from samples at submicromolar concentrations.2 The lack of endo- 1 (Fig. 1) as the first example of a 19F NMR reporter that can
genous fluorine in most biomedical samples eliminates background detect the presence of biologically relevant phosphate anions.12
interference problems, and the wide dispersion of 19F chemical Compound 1 is a zinc(II)-dipicolylamine (ZnDPA) coordination
shifts diminishes the chance of complications due to overlapping complex.13 The organic scaffold is a phenol derivative with two
signals. It is not surprising that 19F NMR is an emerging strategy for ortho-substituted 2,20-dipicolylamine groups and a fluorine
magnetic resonance imaging and is often considered for develop- atom attached to the para carbon. The oxyanion recognition
ment into drug screening assays.3
properties of this class of ZnDPA complexes have been studied
A requirement for essentially all of these 19F NMR techniques is quite extensively, and strong association is typically observed
development of fluorine-labelled reporter molecules. Since many with polyanionic phosphate esters.14 Most notably, optically
drug structures contain fluorine atoms, it is logical to directly study active versions of these compounds with chromophores
fluorine-labelled fragments or drug candidates using 19F NMR appended to the para carbon of the phenol scaffold have been
screening methods that monitor a pharmaceutically relevant protein observed to undergo red-shifts in absorption maxima.14b,d,g
A
binding process.4 In other cases, the 19F label is incorporated within logical explanation for this effect is that phosphate coordina-
the structure of an enzyme substrate and the change in chemical tion to the zinc cations pushes electron density onto the para
shift upon enzyme action is detected as an assay output signal.5 carbon and into the attached chromophore. If true, we reasoned
Fluorine-labelled enzyme substrates have also been incorporated that a fluorine label on the para carbon would exhibit a predictable
into new methods for 19F MRI. The results of enzyme action alter the change in chemical shift upon phosphate binding. Tentative
substrate structure and produce either a change in signal chemical support for this idea was a reported difference in 19F chemical
shift or relaxation time.6 The central concept that is described shifts for the zinc and copper salts of compound 1 (measured in
here is a 19F NMR reporter with ability to undergo a change in the absence of coordinating anions).15
chemical shift upon reversible association with a molecular target.
Structural evidence for our design concept was gained by
elucidating the solid-state complex of reporter 1 bound to
dihydrogen phosphate. We discovered that mixing compound
1 with sodium phosphate in water produced single crystals that
were amenable to analysis by X-ray diffraction.‡ Refinement
of the data produced the molecular structure shown in Fig. 2.
The general coordination features and bond lengths agree with
Department of Chemistry and Biochemistry, University of Notre Dame,
236 Nieuwland Science Hall, Notre Dame, IN, USA. E-mail: smith.115@nd.edu
† Electronic supplementary information (ESI) available: Synthesis, X-ray, and
NMR data. CCDC 929896. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3cc42169d
c
5070 Chem. Commun., 2013, 49, 5070--5072
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Fig. 2 X-ray crystal structure of 1(H2PO4)(NO3)2ꢁH2O. The structure omits the
ꢀ
two NO3 anions for clarity.
related literature structures.16 As expected, the bidentate
dihydrogen phosphate dianion bridges the two zinc cations
in 1. One zinc cation adopts a five-coordinate geometry and the
other is six-coordinate with a water molecule occupying
the additional coordination site. Related crystal structures in
the literature with poylanionic phosphates such as pyrophos-
phate (PPi) or ADP show tetradentate coordination with four
phosphate oxygens coordinated to the two zinc cations.14b,c,f
Together, the X-ray structures suggest that tetradentate coordina-
tion of poylanionic phosphates to compound 1 will push electron
density onto the para carbon and produce an upfield change in
19F chemical shift for the attached fluorine.
Fig. 3 Partial 19F NMR spectra (564 MHz) of reporter 1 (0.40 mM in HEPES
buffer, pH 7.2) with no anion added (bottom spectrum) and in the presence of
one molar equivalent of the following anions as their sodium salts; phosphate
(Pi); pyrophosphate (PPi); ATP; ADP; AMP; o-phospho-L-serine (P-Ser); o-phospho-
L-tyrosine (P-Tyr); citrate.
19F NMR titration studies added incremental amounts of
oxyanions to separate samples of compound 1 (0.40 mM in
50 mM HEPES buffer, pH = 7.2).§ Shown in Fig. 3 are partial 19
F
NMR spectra of 1 in the absence and presence of one molar
equivalent of different oxyanions. The 19F chemical shift for
compound 1 was observed at 21.5 ppm (internal KBF4 as
reference) and there was no detectable signal change produced
by addition of P-Ser, P-Tyr, or citrate. Significant changes in
chemical shift were produced by addition of PPi, ATP, ADP,
phosphate (Pi), and AMP. The anions with high amounts of
negative charge produced relatively large upfield changes in
chemical shift. The slightly downfield effect produced by AMP
is attributed to anisotropic shielding by the proximal adenosine
ring. In the case of ATP binding, a broad 19F signal was observed,
even when 1 was saturated with ATP, indicating an intramolecular
process that exchanges the bound ATP between different
phosphate zinc coordination geometries. As expected, binding
constants with these polyanionic phosphates were too strong
(Ka > 104 Mꢀ1) for accurate determination by NMR.¶ A further
complication preventing NMR measurements of association
was the intermolecular exchange broadening that occurred
when the ratio of anion to 1 was sub-stoichiometric (see ESI†).
Therefore we measured relative anion binding affinities by
conducting competition experiments that mixed reporter 1 with
different ratios of competing anions. Shown in Fig. 4 are
Fig. 4 Partial 19F NMR spectra (564 MHz) of reporter 1 mixed with two
competing anions (as their sodium salts). Each of the three components was
0.40 mM in HEPES buffer, pH 7.2.
selected spectra for samples that contained equal amounts of of reporter molecule 1 in Fig. 5 reflect sequential conversion of
two competing anions. Analysis of all the data indicates that 1 ATP into ADP followed by further hydrolysis and production of
has a relative affinity order of PPi > ADP E ATP > Pi.
Pi. The spectra show clearly that the first step in the process
To demonstrate the potential utility of reporter 1, we used it (hydrolysis of ATP) is complete after 12 minutes, whereas the
to track an enzymatic reaction; namely, the hydrolysis of ATP second step (hydrolysis of ADP) is much slower and takes well
catalysed by commercially available apyrase.17 The 19F spectra over 41 hours. The high ATPase/ADPase ratio was confirmed by
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A typical acquisition collected 1000 scans using a 301 pulse and
0.10 second relaxation delay.
¶ A close structural analogue of 1 is reported to bind PPi and Pi with
association constants of 6.7
ꢃ
106 Mꢀ1 and 1.1
ꢃ
105 Mꢀ1
,
respectively.14h
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independent experiments that started with a sample of pure
ADP (see ESI†) and closely matched the vendor’s certification of
relative enzyme activities. These enzyme hydrolysis experiments
highlight the power of this NMR method to report kinetic
information for sequential steps in the same assay sample. In
contrast, fluorescent probes that can only report single steps, such
as ATP consumption through emission intensity changes, are
unable to provide information about any subsequent chemistry.17
In principle, it should be possible to use compound 1 to monitor
other reactions that consume ATP such as kinase catalyzed
phosphorylation processes. Compound 1 may also be useful in
cell imaging studies of polyphosphate anions. This work was
supported by the NIH (GM059078).
Notes and references
‡ A detailed description of the X-ray structure is provided in the ESI.† 15 S. Torelli, C. Belle, I. Gautier-Luneau, S. Hamman and J. L. Pierre,
The crystallographic data is also provided in CCDC 929896. Inorg. Chim. Acta, 2002, 333, 144–147.
§ Typical 19F NMR samples contained 0.40 mM of 1 in 0.5 mL of 16 E. J. O’Neil, H. Jiang, J. J. Gassensmith and B. D. Smith, Supramol.
50 mM HEPES buffer at 22 1C, pH = 7.2 with an external lock sample Chem., 2013, DOI: 10.1080/10610278.2013.776170.
containing D2O. The internal reference was KBF4 (ꢀ150.4 ppm relative 17 (a) Z. Xu, N. J. Singh, J. Lim, J. Pan, H. N. Kim, S. Park, K. S. Kim and
to CFCl3) and T1 for the 19F signal of free 1 was measured to be
0.59 ꢂ 0.01 second at 22 1C (operating frequency was 564 MHz).
J. Yoon, J. Am. Chem. Soc., 2009, 131, 15528–15533; (b) S. V. Arman
and A. W. Czarnik, Supramol. Chem., 1993, 1, 99–101.
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5072 Chem. Commun., 2013, 49, 5070--5072
This journal is The Royal Society of Chemistry 2013