Long-lived 1H nuclear spin singlet in dimethyl maleate revealed by addition of thiols

Article abstract of DOI:10.1002/anie.201310284

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) have become important techniques in many research areas. One major limitation is the relatively low sensitivity of these methods, which recently has been addressed by hyperpolarization. However, once hyperpolarization is imparted on a molecule, the magnetization typically decays within relatively short times. Singlet states are well isolated from the environment, such that they acquire long lifetimes. We describe herein a model reaction for read-out of a hyperpolarized long-lived state in dimethyl maleate using thiol conjugate addition. This type of reaction could lend itself to monitoring oxidative stress or hypoxia by sensitive detection of thiols. Similar reactions could be used in biosensors or assays that exploit molecular switching. Singlet lifetimes of about 4.7 min for ¹H spins in [D₄]MeOH are seen in this system. Hyperpolarized singlet states are well isolated from the environment, such that they acquire long lifetimes. Here, we used the thiol conjugate addition to unveil the hyperpolarized long-lived singlet state in deuterated dimethyl maleate (see picture). Singlet lifetimes of 4.7 min for ¹H spins in [D₄]MeOH are seen in this system.

Full text of DOI:10.1002/anie.201310284

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Angewandte
Communications
DOI: 10.1002/anie.201310284
Long-Lived Spin States
Long-Lived 1H Nuclear Spin Singlet in Dimethyl Maleate Revealed by
Addition of Thiols**
Yuning Zhang, Pei Che Soon, Alexej Jerschow,* and James W. Canary*
Abstract: Nuclear magnetic resonance (NMR) spectroscopy
and magnetic resonance imaging (MRI) have become impor-
tant techniques in many research areas. One major limitation is
the relatively low sensitivity of these methods, which recently
has been addressed by hyperpolarization. However, once
hyperpolarization is imparted on a molecule, the magnet-
ization typically decays within relatively short times. Singlet
states are well isolated from the environment, such that they
acquire long lifetimes. We describe herein a model reaction for
read-out of a hyperpolarized long-lived state in dimethyl
maleate using thiol conjugate addition. This type of reaction
could lend itself to monitoring oxidative stress or hypoxia by
sensitive detection of thiols. Similar reactions could be used in
biosensors or assays that exploit molecular switching. Singlet
lifetimes of about 4.7 min for 1H spins in [D4]MeOH are seen
in this system.
spin system into an AB or AX spin system by a chemical
reaction) of hyperpolarized ethylene gas by electrophilic
addition with an arenesulfenyl chloride was reported, dem-
onstrating much longer singlet life times for singlet states in
ethylene in the gas phase.[7] Switching in and out of such long-
lived states, in particular singlet or near-singlet states, has
been performed with field-cycling[5,8] and by pulse sequen-
ces.[6b,9] It has also been shown by desymmetrization of 13C-
enriched diacetyl, and a 13C half-life of over 1 min was
reported.[7,10] The very sensitive response to changes in
solvent proved the principle of symmetry-switching, opening
the door to the possibility that a probe could be developed
that would respond to a molecular analyte.
Here we describe a desymmetrization reaction suitable
for unlocking hyperpolarized singlet states in a reaction
inspired by the enzyme maleate isomerase, whose mechanism
of action involves addition of a thiol to maleate.[11] 1H
lifetimes of up to 4.7 min in [D4]MeOH were achieved (a 24-
fold enhancement over T1). We also discuss the relaxation
mechanisms that limit the lifetimes of these states.
H
yperpolarization has become a very active research area
in NMR spectroscopy and MRI because of the prospect of
sensitivity enhancements[1] on the order of 102–105. One long-
term goal is the establishment of contrast agents that can,
after sensitivity enhancement, be injected in the body and
later measured independently of the weaker background
signals.[2] Other areas of application may be the monitoring of
chemical reactors,[3] or the use of biosensors, such as Xe-
biosensors.[4] One sticking point in many of these proposals is
the lifetime (on the order of T1 within 20 s) of the magnet-
ization states that are imparted on the molecules. The short
lifetimes can be circumvented by the use of long-lived states,
which can be based on nuclear singlet states (or other
relaxation-minimized subspaces). For 1H spins in solution
a record was achieved of a 4 min half-life,[5] while longer times
are technically possible for low-gamma nuclei generally due
to weaker relaxation mechanisms.[6] While this manuscript
was in preparation, desymmetrization (conversion of an A2
The symmetry switch strategy is shown in Figure 1a where
hydrogenation of dimethyl acetylene dicarboxylate (DMAD;
1) with parahydrogen yields hyperpolarized dimethyl maleate
(DMM; 2). Due to magnetic equivalence, the nuclear singlet
state of 2 is preserved and remains invisible to NMR
[*] Y. Zhang,[+] P. C. Soon,[+] Prof. A. Jerschow, Prof. J. W. Canary
Department of Chemistry, New York University
100 Washington Sq. East, New York, NY 10003 (USA)
E-mail: alexej.jerschow@nyu.edu
james.canary@nyu.edu
Homepage: http://www.nyu.edu/projects/jerschow/
http://www.nyu.edu/pages/canary/home.html
Figure 1. Revelation of long-lived nuclear spin singlet in compound 2
by desymmetrization: a) Reaction scheme. b) 1H OPSY spectrum of
hyperpolarized 4c. c) Thermal spectrum of 4c after the hyperpolariza-
tion decayed. “S” denotes solvent peak. The relative stereochemistry of
the protons in the major (70%) and minor (30%) products was
derived from the splitting pattern of the peaks and the coupling
constants. The peak corresponding to Hc* is a doublet with a large
coupling constant (3J=10 Hz), indicating a dihedral angle between
Hc* and Ha* of about 1808. Hb* is broadened because the dihedral
angle between Hb* and Ha* is about 608, and thus, the fine splitting
pattern due to a small coupling constant was not resolved.
[+] These authors contributed equally to this work.
[**] This work was supported by NSF grants CHE-0848234 (J.C.) and
CHE-0957586 (A.J.). The experiments were performed in the Shared
Instrument Facility of the Department of Chemistry, New York
University, supported by the US National Science Foundation under
Grant No. CHE0116222.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310284.
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Chemie
detection.[5] To access this long-lived nuclear singlet state, we
used a facile base-catalyzed thiol addition reaction with 2. In
the product, 4, the two hyperpolarized hydrogen atoms are
magnetically inequivalent, and so, readily convert to the
triplet state, which is observed by an only parahydrogen
spectroscopy (OPSY) pulse sequence.[12] The OPSY spectrum
in Figure 1b shows the new anti-phase peaks characteristic of
parahydrogen induced polarization (PHIP). The addition of
thiols to 2 is known to be diastereoselective[13] leading to the
appearance of hyperpolarized protons Hb* (major) and Hc*
(minor) and resulting in additional line splitting.
Figure 2. Pulse sequence and the magnetic field path experienced by
We adapted the hydrogenation reaction of 1 to proceed in
deuterated methanol (rather than in acetone),[5] since base-
catalyzed nucleophilic addition of aliphatic thiols to 2 is
sluggish in acetone. The choice of a protic solvent is also
a better intermediate step towards an eventual goal of
performing water-based reactions. When 1 was hydrogenated
with parahydrogen at earth field and then transported to the
observation field (Figure 2, path A), the hyperpolarized
signals due to nuclear triplet states decayed within 2 min
(Figure S1 in the Supporting Information). The hydrogena-
tion reaction was found to be completed within 30 s (Fig-
ure S2).
the sample. At earth field, 1 was hydrogenated with parahydrogen to
yield 2. The NMR sample tube, along with the syringe containing the
thiol solution, was inserted into the magnet (11.7 T) following path A
(blue). Subsequently, a 908 pulse was used to spoil the signal resulting
from transportation across different magnetic fields. After waiting for
time Tw, the thiol solution was injected into the NMR tube to
desymmetrize 2, yielding 4 and the hyperpolarized signals for Ha*, Hb*
and Hc* observed using the OPSY sequence. The OPSY spectrum is
displayed beside its free induction decay (FID). In another set of
experiments, the hydrogenated sample was left at earth field for
various waiting times Tw, then transported to observation field for thiol
addition (path B, green).
For singlet state read-out, we implemented the thiol
addition reaction. This reaction could potentially be further
developed into an enzyme-responsive system. Mercaptoetha-
nol (3a) and its deuterated analog (3b) were used for base-
catalyzed thiol addition experiments.[14] The reaction between
2a and 3a was facile and the half-life was estimated to be 12 s
from following the consumption rate of 2a by UV/Vis
spectroscopy. In order to inject the thiol solution into the
NMR sample tube in the spectrometer, we
18 s respectively. One can deduce that the thiol reaction is
complete (Figure S4c) after one minute. Hydrolysis of com-
pounds 2a and 4a was not observed (Figure S4c). For 4a and
4b, data is only available for Hc, which is isolated in the
thermal spectrum. As a next step, we implemented the
reaction with a biologically relevant thiol (cysteine) as the
chemical trigger (Figure 4).
inserted a segment of a long tubing through
a septum cap into the NMR sample solution. The
tubing was connected to a syringe containing the
thiol solution.[15] A typical experiment proceeded
in the following manner (Figure 2): After hydro-
genation of 1 with parahydrogen at earth field, we
transported the NMR sample tube, along with the
tubing, to the observation field and applied
a spoiler pulse. After several minutes (Tw), the
thiol solution was injected simultaneously with
the initiation of the OPSY pulse sequence.
To ensure that hydrogenation of 1 did not re-
initiate upon thiol addition (the average yield of 2
was 80% with 20% of unreacted 1), we injected
the thiol solution prior to hydrogenation with
parahydrogen. No hyperpolarized peaks or for-
mation of 2 were observed because 3a added
readily to 1, making it unavailable for hydro-
genation (Figure S3).
We plotted the 1H OPSY signal and enhance-
1
Figure 3. Decay of long-lived singlet state unveiled by chemical switching: H OPSY
signal (normalized to the thermal peak, shown on the left y-axis) and enhancement
factor (right y-axis) of hyperpolarized 1H decays as a function of Tw at 11.7 T (a, b, c)
ment factor (EF) of individual hyperpolarized
protons as a function of Tw (Figure 3). In consec-
utive OPSY experiments, the persistent hyper-
polarized signals detected for at least a minute
arise from the ongoing thiol reaction. In 4a, T1 of
Hc is 12 s while for 4c, T1 values of Ha, Hb and Hc
~
&
*
or at earth field (d). EF (bigger symbols) of Ha ( ), Hc ( ) and Hb ( ) are fitted to an
exponential decay to determine Ts. a) Hc of 4a: Ts of 2a at 11.7 T is 2.45Æ0.61 min;
b) Hc of 4b: Ts of 2b at 11.7 T is 4.65Æ0.95 min; c) Ha, Hb and Hc of 4c: Ts of 2b at
11.7 T is 4.69Æ0.37 min; d) Ha, Hb and Hc of 4c: Ts of 2b at earth field is
are 21 s (average of two diastereomers), 14 s and 3.39Æ0.47 min.
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11.7 T. This trend indicates that the residual relaxation
mechanisms are in a relatively fast motion regime.
Signal-revealing chemical reactions are desirable for their
ability to detect certain molecular events. Many different
molecular switches have been reported, including those that
respond to electromagnetic field, oxidation potential, chem-
ical reaction, and others.[19] Diverse properties have been
explored as read-out for molecular switches, especially
optical, magnetic, and electronic effects.[20] Environment-
sensitive MRI probes have attracted significant interest in
order to take advantage of the deep-tissue access afforded by
MRI to image the expression of molecular phenomena
associated with normal or pathogenic physiology or metab-
olism.[20c,21] A few examples of MRI contrast agents that
demonstrate differential relaxivity in the presence of an
environmental change have been reported,[21,22] but no
example has been reported of a probe that shows a turn-on
spectroscopic response to a biologically relevant stimulus.
In conclusion, nuclear singlet order created by PHIP was
stored in a chemically symmetrical molecule at high field with
half-lives of up to 4.7 min. Hyperpolarization was unveiled by
a chemical reaction with thiols. Thiols represent a particularly
useful analyte, opening prospects for responsive and biosen-
sor systems with environmentally induced NMR spectroscop-
ic signal turn-on mechanisms.
Figure 4. Decay of the long-lived singlet state unveiled by adding
cysteine: a) 1H OPSY signal (normalized to the thermal peak, shown
on the left y-axis) and EF (right y-axis) of hyperpolarized 1H decays as
a function Tw at 11.7 T. Data for Ha is unavailable because the peak is
~
*
unresolved in the thermal spectrum. EF of Hc ( ) and Hb ( ) were
fitted to an exponential decay to determine the Ts of 2b at 11.7 T to be
4.69Æ0.45 min. b) 1H OPSY spectrum of hyperpolarized 4d. c) Ther-
mal spectrum of 4d after the hyperpolarization decayed. “S” denotes
solvent peak, “R” denotes unreacted cysteine peaks, “C” denotes
catalyst peaks.
The lifetime of the singlet state, Ts, of 2a at 11.7 T was
found to be 2.45 Æ 0.61 min (Figure 3a) in both the thiol
addition and the field cycling[5] (Figure S5) procedures. We
expected a somewhat shorter lifetime in [D4]MeOH as
compared to [D6]acetone as used in Ref. [5] (4 min at 7 T).
In solvents that contain hydrogen bonds (even deuterated
ones) such as [D4]MeOH, singlet lifetimes have been reported
to be much shorter as compared to aprotic solvents.[16]
Deuterated DMM (2b) has a longer Ts, 4.65 Æ 0.95 min
(Figure 3b), because of the reduced number of significant
coupling partners.[17] It was verified that field cycling did not
work for 2b (Figure S6b), due to magnetic equivalence in this
compound. Technically, long-range coupling to deuterium
introduces a tiny degree of magnetic inequivalence in the
system. However, due to the weakness of such coupling
constants, field-cycling would produce extremely weak sin-
glet-to-triplet conversion, and/or would require a long resi-
dence time at the resonance field in order for an appreciable
amount to be transferred, something that is not achievable or
desired in practice.
It is interesting to note that the Ts of 2a is 2.45 Æ 0.61 min
while Ts of 2b is 4.65 Æ 0.95 min even though the T1 values of
the vinylene protons in 2a and 2b are both about 21 s. This
trend showed that deuteration had a minimal effect on T1
while singlet relaxation was strongly affected. Intrapair
dipole–dipole (DD) coupling makes a strong contribution to
T1 relaxation,[18] but is largely ineffective for singlet relaxa-
tion. Other contributions to relaxation, such as out-of-pair
DD coupling, can then form the residual relaxation mecha-
nisms that limit Ts. With deuteration of the methyl group, the
out-of-pair DD coupling is reduced so that the Ts is longer.
Chemical shift anisotropy (CSA) and spin-rotation relaxation
could be additional lifetime limiting factors.[6b]
Received: November 27, 2013
Revised: January 28, 2014
Keywords: cysteine · long-lived spin states ·
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NMR spectroscopy · parahydrogen · thiol conjugate addition
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