Detection of picomole amounts of biological substrates by para-hydrogen-enhanced NMR methods in conjunction with a suitable receptor complex

Article abstract of DOI:10.1021/ja074286k

NMR monitoring of solutions containing para-hydrogen and an iridium-based reporter complex is shown to allow the detection of picomole quantities of biological substrates containing suitable donor sites. Copyright

Full text of DOI:10.1021/ja074286k

Published on Web 08/21/2007
Detection of Picomole Amounts of Biological Substrates by
para-Hydrogen-Enhanced NMR Methods in Conjunction with a Suitable
Receptor Complex
Nicholas J. Wood, James A. Brannigan, Simon B. Duckett,* Sarah L. Heath, and Jane Wagstaff†
Department of Chemistry, UniVersity of York, Heslington, YO10 5DD, York, U.K., and Department of Chemistry,
UniVersity of Manchester, Oxford Road, Manchester M13 9PL, U.K.
†
†
,†
‡
Received June 13, 2007; E-mail: sbd3@york.ac.uk
1
Since the discovery of the anti-cancer drug cis-platin, and the
subsequent accumulation of evidence regarding its mode of action,2
the reaction chemistry of transition metals toward biological ligands
3
such as DNA nucleotides has been the focus of intense study. Of
particular interest is the mode of binding that these ligands exhibit;
while DNA itself offers a multiplicity of N- and O-donor sites, it
is generally accepted that the remarkable efficacy of cis-platin arises
from its ability to form intrastrand cross-links, by binding covalently
7
4
to the N sites in guanine. Previous studies have shown the para-
hydrogen enhancement phenomenon (PHIP) to be a valuable tool
5
in the study of species formed via reactions with hydrogen. Here,
we use PHIP to detect picomole amounts of biological substrates
by reference to the binding of pyridine, purine, and adenine to IrCl-
1
Figure 1. Hydride region of H NMR spectra obtained from a sample of
1
5
IrCl(CO)(PPh3)2 and p-H2 at 325 K containing (a) pyridine and (b) N-
pyridine (now P decoupled).
2 3 2
(H) (PPh ) . This approach provides a sensitivity gain that exceeds
31
that of an NMR cryo-probe by at least 2 orders of magnitude.
Optimized detection of DNA base moieties by tip-enhanced Raman
Scheme 1. Formation of IrCl(H)2(PPh3)2(pyridine) 1
6
7
methods and HPLC-MS offer similar detection limits.
For this work, NMR tubes were charged with ∼0.9 µmol (0.7
mg) of IrCl(CO)(PPh , and the biological ligand, 0.12 mmol (10
µL) of pyridine (py), and made up with d -toluene. Samples were
placed under 3 atm of pure p-H , shaken, and then introduced into
3 2
)
8
8.46, thereby demonstrating the spatial proximity of the hydride
2
ligand to both the phosphine ligand and the ortho proton of pyridine.
Key NMR data for 1 can be found in the Supporting Information.
This complex has been partially characterized previously.
To learn about the methods sensitivity to detecting pyridine
appropriate samples were prepared. After 32 scans at 325 K and
1
the spectrometer at 295 K. The H NMR spectra obtained under
these conditions showed enhanced hydride resonances at δ -6.86
9
8
and -17.58 due to IrCl(CO)(H)
2
(PPh
3
)
2
. At 325 K, two new
mutually coupled hydride signals due to 1 were observed at δ
21.03 and -21.82 (Figure 1). Each of these signals appears as a
triplet of antiphase doublets where the antiphase component arises
from coupling between the two former p-H nuclei. The triplet
couplings vanish with phosphorus decoupling, and when a H-
-
4
00 MHz (∼1 min of acquisition), a signal-to-noise ratio of 11
was obtained for the hydride resonances of 1 when the pyridine
concentration was 0.1 µM, diagnostic of 50 pmol of substrate.
To test the response of the system to a more sterically demanding
substrate, pyridine was replaced by benzimidazole. In the associated
2
1
31
P HMQC experiment was recorded, both hydrides’ resonances
connected to a single 31P signal at δ 21.75. These data are consistent
with a structure of 1 containing two trans phosphine ligands are
arranged cis to two inequivalent hydride ligands.
1
H NMR spectra, hydride signals due to 2, the benzimidazole ana-
1
5
logue of 1, could be seen at δ -19.22 and -22.19. The N chem-
ical shift of the coordinated nitrogen atom of the five-membered
ring of 2 was located on a non-enriched sample at δ -137.
13
Upon repetition of the experiment using CO-labeled IrCl(CO)-
PPh
13C-derived couplings to the hydride signals were observed
for IrCl(H) (CO)(PPh , but not for 1, demonstrating the absence
(
3 2
) ,
The IrCl(CO)(PPh
purine. An NMR tube containing p-H
IrCl(CO)(PPh yielded six new hydride signals at 305 K due to
three products, 3a-c, isomers of IrCl(H) (purine)(PPh . The
3
)
2
system was then tested as a sensor for
2
3 2
)
2
, 4.2 µmol of purine, and
of a carbonyl ligand in the new species. Repeating the experiment
with 0.03 mmol (2.5 µL) of 15N-labeled pyridine gave rise to an
extra 17.7 Hz coupling in the hydride resonance at δ -21.03,
consistent with it arising from a proton trans to pyridine. Product
3 2
)
2
3 2
)
hydride resonances for 3a (δ -21.28 and -22.70) are of a much
greater magnitude than those for 3b (δ -20.86 and -21.82), which
are themselves of slightly greater magnitude than those for 3c (δ
1
15
1
2 3 2
is therefore IrCl(H) (PPh ) (py) (Scheme 1). H- N HMQC
15
experiments allowed the N resonance to be located at δ -131
via magnetization transfer from both hydride ligands. Further
evidence for the pyridine ligand was obtained by 1D NOE
spectroscopy: irradiating the hydride at δ -21.03 yielded an NOE
connection to a δ 7.86 signal corresponding to an ortho-phenyl
proton of triphenylphosphine. In contrast, irradiation of the hydride
at δ -21.82 yielded NOE connections to signals at δ 7.86 and
-
19.57 and -21.86); assuming that each product is enhanced to
the same degree, integration of the resonances indicates that 3a,
3b, and 3c are formed in an approximate ratio of 100:18.6:0.8 at
305 K. Figure 2 illustrates the corresponding spectrum at 325 K.
The chemical shifts and coupling parameters of these products
closely match those of 1 and 2, indicating that each of the new
species is formed from purine ligating through a nitrogen atom;
1
1
these assignments were confirmed using a range of H- H COSY
†
University of York.
University of Manchester.
‡
1
31
and H- P HMQC experiments.
11012
9
J. AM. CHEM. SOC. 2007, 129, 11012-11013
10.1021/ja074286k CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
1
3
9
7
Scheme 3. Structures of the N -, N -, N -, and N -Coordinated
Purine Complexes 3a, 3b, 3c, and 3d
1
31
Figure 2. Hydride region of the H{ P} NMR spectrum of a sample of
IrCl(CO)(PPh3)2 containing 0.5 mg of purine and p-H2 at 325 K. The signal
marked with / is due to IrCl(H)2(PPh3)3.
Scheme 2. Tautomers of Purine and Adenine
sterically hindered site. For 3c, the δ -21.86 hydride signal exhibits
one NOE connection to a ligated purine resonance at δ 8.45. Since
8
the H proton usually resonates at approximately δ 8.7, it is evident
that this signal has also been shifted approximately 0.3 ppm upfield
upon coordination. However, the assignment of this signal to the
8
7
H proton does not distinguish between the N -ligated product and
9
the N -ligated product since both would be expected to show an
NOE to H . In this case, the absence of another NOE interaction
in the spectrum due to H allows 3c to be assigned as containing
an N -ligated purine ligand. Unfortunately, the hydride signals of
8
6
9
Scheme 2 shows the principal tautomers of purine with theoretical
studies suggesting that the N -H tautomer is most stable.
9
10
3d are of insufficient magnitude to conduct NOESY experiments;
however, the δ -19.43 chemical shift of the trans-to-nitrogen
hydride signal, along with the elimination of the other N-donor
sites, allows this species to be identified as the N -coordinated
2 3 2
isomer of IrCl(H) (purine)(PPh ) .
The ligand arrangements in products 3a-d (Scheme 3) were
1
partially assigned by comparison of their H NMR data with that
7
of 1 and 2 since inspection of the chemical shifts of the trans-to-
nitrogen hydride signals shows that this parameter is diagnostic of
the type of heterocyclic ring in which the ligating nitrogen is
situated: hydride signals in the region δ -19.2 to -19.8 arise from
coordination via a five-membered ring, whereas those at δ -21.0
to -21.5 arise from coordination via a six-membered ring. In the
case of 3a-d, it is therefore evident that 3a and 3b are species
where the purine ligand is bound via the pyrimidine moiety, while
When the analogous reaction with adenine was examined,
the resultant 1H NMR spectrum (325 K) contained dominant
PHIP-enhanced hydride signals arising from 4a, at δ -21.37 and
δ -22.74, for 4b at δ -20.90 and for 4c at δ -20.69 and δ -22.22.
1
3
9
These are due to the N -, N -, and N -linkage isomers, respectively.
3
Coordination of the N site of adenine as found in 4b has only
previously been reported when the other sites are blocked.
In this report, we establish that p-H -based NMR methods in
1
1
3
c and 3d contain purine bound via the imidazole moiety. The
binding mode of the purine ligand was further determined by NOE
spectroscopy where connections for the resonance of the hydride
trans to chloride provided the crucial information. This was further
2
conjunction with a suitable reporter complex can be used to detect
picomole quantities of nucleobases via the detection of diagnostic
signals in a normally unoccupied region of the proton NMR
spectrum. The sensitivity of the PHIP-enhanced method compares
favorably with HPLC-MS for the detection of such species.
1
supported by comparison of the H chemical shifts of the
coordinated purine with those of free purine. For 3a, the δ -22.66
hydride signal shows two NOE interactions to purine ligand protons
which resonate at δ 8.98 and 8.65; in 3a the purine is bound via
Supporting Information Available: Synthetic details and key
NMR observations. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
N because this is the only binding site which has two adjacent
6
2
protons (H and H ). In free purine, these protons usually resonate
(
in d -DMSO) at δ 9.21 and 8.99, respectively; it appears that, upon
6
References
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approximately 0.3 ppm relative to the free purine. The N site of
purine is the most basic and least sterically hindered nitrogen donor
site in the molecule, which is consistent with the formation of the
N -ligated product 3a as the most abundant product. The PHIP
attained in the case of 3a proved to be great enough to allow the
rapid location of a N signal at δ -177 for the coordinated N1
nitrogen with the nucleus at natural abundance, which means that
signals equating to the detection of femtomole quantities are seen
when signal averaging is employed. For 3b, although the signals
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shows one NOE interaction arising from protons on the purine
ligand, at δ 8.64. This supports the assignment of 3b as a product
arising from N coordination of purine, as only the H proton is
adjacent to the binding site; again, coordination shifts the H2
resonance approximately 0.3 ppm upfield from its free ligand value.
The relatively small magnitude of the hydride signals of 3b are
1
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3
consistent with this as the N position is the least basic and most
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J. AM. CHEM. SOC.
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