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Published on the web July 24, 2010
Turn-on Detection of Targeted Biochemical Reactions
by Triple Resonance NMR Analysis Using Isotope-labeled Probe
Keigo Mizusawa,1,# Ryuji Igarashi,2,# Kosei Uehira,1 Yoshimasa Takafuji,3 Yasuhiko Tabata,3
Hidehito Tochio,2 Masahiro Shirakawa,*2 Shinsuke Sando,*1,³ and Yasuhiro Aoyama*1,³³
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510
2Department of Molecular Engineering, Graduate School of Engineering, Kyoto University,
Katsura, Nishikyo-ku, Kyoto 615-8510
3Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences,
Kyoto University, 53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507
(Received May 19, 2010; CL-100482; E-mail: shirakawa@moleng.kyoto-u.ac.jp,
ssando@ifrc.kyushu-u.ac.jp, yaoyama@mail.doshisha.ac.jp)
We report on a strategy for the “turn-on” detection of target
The triple resonance technique is a method to correlate three
biochemical (metabolic) reactions using a triple resonance NMR
technique with an isotope-labeled probe. Our NMR study clearly
reveals that otherwise NMR-nonactive-13C/2H-labeled glucose
actually turns “on” its 1H NMR signal by conversion to an
important biomarker lactate as the end product of anaerobic
glycolysis in cells and in injected mice with high selectivity.
NMR-active nuclei with different Lamor frequencies. For
example, when the pulse scheme allows the magnetic coherence
of 1H to transfer to two successive 13C nuclei with different
Lamor frequencies through scalar couplings, only the proton in
the particular sequence 1H-13C-13C is detectable. Due to the low
natural abundance of 13C (1.1%), the probability of a naturally
1
occurring H-13C-13C sequence is as low as 0.01%, suggesting
that the triple resonance technique can markedly improve the
1
There has been a long-term effort into the analysis of
specific chemical events in complex biological systems, and
metabolic profiling is one of the most intriguing targets among a
variety of events. Metabolites are chemical products resulting
from essential biological activities and, therefore, can be good
biomarkers to reveal the physiological or chemical status of
cells, tissues, and organs.
selectivity of detection of a target molecule having H-13C-13C
by lowering the background signal.15
As a model metabolic reaction for demonstrating the proof-
of-concept of our idea, we chose the glucose-to-lactate produc-
tion. Among the stable isotopomers for metabolic analysis by
NMR, 13C-labeled glucose is one of the most intensively
studied. Glucose is a common source of energy in cells and is
converted to a variety of metabolites during glycolysis. Among
these, lactate H3C-CH(OH)-CO2H is a key end metabolite. In
particular, in tumor cells, glucose is actively converted to lactate
through an anaerobic glycolytic pathway.16,17 We challenged to
develop a method to detect generation of lactate from glucose
using isotope-enriched glucose as a probe that turns from “off”
to “on” its own 1H NMR signals in synchronization with a target
anaerobic glycolytic reaction (Figure 1a).
NMR/MRI-based technology is one of the most promising
techniques for the analysis of such biochemical (metabolic)
reactions. However, the detection of target metabolites or
biochemical reactions of interest is often difficult because of
spectral overlaps with a large number of biological components.
To overcome this, attempts to trace metabolic pathways by the
administration of stable isotope-labeled compounds as probes
has been reported.1 In principle, these isotope-labeled com-
pounds are highly potential probes since these are NMR-
sensitivite and constitutively NMR-active. However, this ad-
vantage can also be a disadvantage since, for the same reason,
these probes and derived various intermediates/metabolites all
serve as possible origins of background noise signals, and thus
target metabolite should be carefully discriminated from others
by the precise analysis of the chemical shifts, typically by
measuring the 2D NMR spectra.1,2 Especially, such spectral
overlaps are problem for detection of 1H, the most sensitive and
thus attractive nucleus, due to its small chemical shift range.
In this context, ideal chemical probes are those with a signal
on/off device, that are otherwise NMR-silent and is rendered
NMR-active only when accumulated at target sites and subjected
to specific biochemical events.3-14 In this work, we have used a
triple resonance NMR technique for the detection of isotopically
labeled metabolites generated in cells and in injected mice with
high selectivity. In addition, for the first time, we report on
a signal-activatable glucose-based probe, which turns “on”
its 1H NMR signals as a result of target anaerobic glycolytic
reaction.
First, we measured a triple resonance NMR spectrum of
chemically synthesized 13C-labeled lactate. We used a pulse
sequence for the detection of a H bound to an aliphatic 13C,
1
which in turn was connected to a carbonyl 13C (1H-{13C-
13C¤}).18 The conventional 1H NMR spectrum of 1,2,3-13C-
labeled racemic lactate, prepared by chemical reduction of 1,2,3-
13C-labeled pyruvate using NaBH4,19 in D2O shows two double
multiplets attributable to methine protons (2-C, 4.21 ppm,
J
C-H = 147 Hz) and methyl protons (3-C, 1.30 ppm, JC-H =
129 Hz) (Figure S1a21). In marked contrast, only methine
protons (2-C) were detected in the triple resonance experiment
(Figure S1b21) with a detection limit of 77 ¯M under our
experimental conditions (data not shown).
Once the triple resonance technique was optimized to detect
targeted lactate, we moved on to sensing of the biomarker lactate
using isotope-labeled glucose as a precursor probe. Initially, we
used the glucose probe 1 (Figure 1a), where all the carbon atoms
were enriched with 13C. Again, our purpose was to develop an
OFF-to-ON-type metabolic sensing probe that would ideally be
undetected until subjected to the metabolic reaction of concern.
Chem. Lett. 2010, 39, 926-928
© 2010 The Chemical Society of Japan