This is the peer reviewed version of the following article:
Application of Hydrogen Isotopes in the Life Sciences, J. Atzrodt, V. Derdau, W.J. Kerr and M.
Reid, Angew. Chem. Int. Ed., 2017, doi: 10.1002/anie.201704146
which has been published in final form at:
http://onlinelibrary.wiley.com/doi/10.1002/anie.201704146/abstract
This article may be used for non-commercial purposes in accordance With Wiley-VCH Terms
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Applications of Hydrogen Isotopes
in the Life Sciences
Jens Atzrodt,
[a],
Volker Derdau,
[a],
William J. Kerr,
[b]
and Marc Reid
[b].
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3
Abstract: Hydrogen isotopes are unique tools for identifying and
understanding biological or chemical processes. Hydrogen
isotope labeling allows for a traceless and direct incorporation of
an additional mass or radioactive tag into an organic molecule
with almost no change in its chemical structure, physical
properties or biological activity. Using deuterium labeled
isotopologues to study the unique mass spectrometric (MS)-
pattern generated from mixtures of biological relevant molecules
drastically simplifies analysis. Such methods are now providing
unprecedented levels of insight in a wide and continuously
growing range of applications in the life sciences and beyond.
Tritium (
3
H), in particular, has seen an increased utilization,
especially in pharmaceutical drug discovery. The efforts and
costs required for the synthesis of labeled compounds are more
than compensated for by the enhanced molecular sensitivity for
analysis and high reliability of the data obtained. In this review,
advances in the applications of hydrogen isotopes in the life
sciences are described.
Jens Atzrodt studied chemistry in
Jena and obtained his PhD in 1999
with Prof. R. Beckert before joining
Aventis Pharma Germany (today
Sanofi) as laboratory head and later
as section head in the Medicinal
Chemistry department (Sanofi)
responsible for Isotope Chemistry &
Metabolite Synthesis (ICMS). Today
he is the head of the Hub
Management Office at Sanofi Frankfurt. From 2018 he will
become President of the International Isotope Society and is
presently a member of the advisory board of the Journal of
Labeled Compounds and Radiopharmaceuticals (Wiley).
Volker Derdau studied chemistry in
Münster and Braunschweig and
obtained his PhD in 1999 with Prof.
Sabine Laschat. He went for a one
year DAAD funded Post-Doc in Prof.
Victor Snieckus group (Kingston,
Canada) before he started at Aventis
Pharma Germany (today Sanofi) as
laboratory head. Today he is section
head in the Medicinal Chemistry
department (Sanofi) responsible for Isotope Chemistry &
Metabolite Synthesis (ICMS), senior lecturer at the University
of Applied Sciences (Darmstadt), and European Associate
Editor of the Journal of Labeled Compounds and
Radiopharmaceuticals (Wiley).
Billy Kerr studied at the University of
Strathclyde, gaining his PhD in 1986
with Prof. Peter Pauson and Dr
David Billington. Following
postdoctoral research at Brandeis
University (Waltham, MA, USA) with
Prof. Myron Rosenblum, and
Imperial College (London, UK) with
Professor Steven Ley, he returned
to Strathclyde in 1989 as a Lecturer
in Organic Chemistry. Following
Senior Lectureship, he was promoted to a Professorial Chair in
2002. He now holds the endowed Chair of 1919 Professor of
Organic Chemistry and is Deputy Associate Principal at
Strathclyde. He was elected as a Fellow of the Royal Society
of Edinburgh (FRSE) in 2014, and in 2015 was the recipient of
the Melvin Calvin Award for outstanding contributions to
Isotope Chemistry.
Marc Reid earned his PhD (2015) in
organic and computational chemistry
(w/ Prof. William J. Kerr and Dr Tell
Tuttle) from the University of
Strathclyde. Recently, he completed
postdoctoral studies at the University
of Edinburgh (w/ Prof. Guy Lloyd-
Jones, FRS). In 2016, he was
selected for the SciFinder Future
Leaders program, and in 2017, he
was selected to be among the Young Observers for the IUPAC
World Chemistry Leadership Meeting. Marc is currently
pursuing independent research at Strathclyde as a
Leverhulme Fellow and GSK-funded Early Career Academic,
where his research interests include transition metal catalysis,
kinetics, and cheminformatics.
[a] Dr. J. Atzrodt, Dr. V. Derdau
Isotope Chemistry and Metabolite Synthesis, Integrated Drug
Discovery, Medicinal Chemistry,
Industriepark Höchst, G876, 65926 Frankfurt
E-mail: Jens.Atzrodt@sanofi.com; Volker.Derdau@sanofi.com
[b] Prof Dr W. J. Kerr, Dr M. Reid
Department of Pure and Applied Chemistry, WestCHEM,
University of Strathclyde, 295 Cathedral Street,
Glasgow, Scotland, G1 1XL (U.K).
E-mail: w.kerr@strath.ac.uk; marc.reid.100@strath.ac.uk
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1. Introduction
For many years, hydrogen isotopes (deuterium and tritium)
have been known for their utility in mechanistic, spectrometric,
and tracer studies. Moreover, well known applications of
hydrogen isotopes exist within almost every sub-discipline in life
science, in nuclear science, and beyond.
[
1
]
Today, the ability for
precise measurement of isotope ratios promotes a dynamic view
on biosynthetic pathways, protein turnover, and systems-wide
metabolic networks and, thus, has paved the way for a number
of scientific breakthroughs in biomedical research.
[
2
]
In medicinal
chemistry, replacement of hydrogen by deuterium has recently
received much attention as a way to alter absorption, distribution,
metabolism, and excretion (ADME) properties of drug
candidates.
[
3
]
Thus, the objective of this review is to provide a brief
perspective on the rapidly increasing applications of hydrogen
isotopes in life science (Scheme 1).
Scheme 1: Applications of hydrogen isotopes in different areas of the Life
Sciences
Hydrogen isotopes have many properties of ideal tracer
nuclides. Both deuterium and tritium can be detected with very
high sensitivity, applying conventional mass spectrometry for the
former or radioactivity measurements for the latter. In recent
years, the rapid development of high performance mass
spectrometry has increased deuterium labeling applications
significantly, while tritium continues to play a flagship role in drug
discovery.
[
4
]
The popularity of hydrogen isotopes in the life
sciences stems from their ability to allow for direct incorporation
of a unique detection signal into the target molecule without
changing its chemical structure, physical properties or biological
activity. Consequently, hydrogen isotopes enable the detection
and quantification of drug-related material or the discovery of
new biological pathways in systems as complex as experimental
animals or humans.
[
5
]
Deuterium is a stable isotope and thus can be handled under
standard wet lab conditions without special permission, handling
licenses, or radiation safety measures. This is not the case for
tritium. Having said this, emissions are weak enough to make
only minimum shielding necessary. Due to the long half-life (12.3
years) of tritium, it is unnecessary to correct for decay during
analysis. Thus, once prepared
3
H-tracer can be stored and used
for a long period of time if radiolytical decomposition can be
minimised.
Compared to
13
C or
14
C, hydrogen isotope labeling is
typically easier, quicker, and much cheaper.
[
6
]
On the other hand,
it is more difficult to predict the metabolic stability of
2
H or
3
H
labeled compounds. In case of tritium, a reduced biologically
tracer stability may result in an in vivo formation of highly toxic
3
H
2
O which can be distributed throughout the whole body and
thus makes radioactivity measurement and quantification more
difficult.
[
7
]
Incorporation of deuterium or tritium into an organic molecule
can be achieved by two principle routes, either by a conventional
multistep synthesis or by direct hydrogen isotope exchange
(HIE). Depending on the complexity of the chemistry, the
chemical structure of the target molecule, and the labeling
position, a classical synthesis approach, starting from
appropriate commercially available labeled precursors, can be
very time and resource consuming. Therefore, methods for fast
and convenient late stage introduction of deuterium or tritium
into organic molecules were extensively investigated in recent
years. The hydrogen isotope exchange (HIE)
[
8
,
9
]
reaction allows
for a selective installation of C–D
[
10
]
and C–T
[
11
]
bonds in the
target molecule. Typically heterogeneous metal-catalyzed HIE
generally results in relatively unspecific incorporation of
numerous deuterium atoms into a molecular substrate.
Accordingly, heterogeneous metal-catalyzed H/D exchange is
typically the method of choice for preparation of stable
isotopically labeled internal standards (SILS) for LC-MS/MS
investigations. For SILS applications, the similarities of the mass
signals for the unlabeled analyte relative to the signals for the
internal standard should be as low as reasonably feasible. As a
consequence, heterogeneous exchange methods have been
optimized to incorporate 3 – 5 deuterium atoms in the case of
small molecules without chlorine, bromine, or sulfur-containing
functionalities and where the remaining amount of unlabeled
(D
0
) is negligible.
[
12
]
In contrast, homogeneous metal catalyzed HIE methods are
typically much more selective incorporating deuterium only at
specific positions in the molecule, e.g. next to a directing group.
Therefore, these methods are of particular importance for tritium
incorporation via H/T exchange
[11]
The breadth of applications
and molecular structures that require isotopic labeling ultimately
demand a suite of synthetic methods in order to install the
isotope in the desired position(s). These methods are
highlighted in the connected review “C-H Functionalization for
Hydrogen Isotope Exchange”.
[
13
]
2. Applications of Deuterium labeled
compounds
Applications of deuterium can be distinguished based on
four general concepts: 1. kinetic and equilibrium isotope effects;
2. the generation of specific MS-patterns (e.g. 1:1 mixtures of
labeled and unlabeled analyte); 3. the utilization for relative
quantification based on changes of the ratio of labeled versus
unlabeled analyte; and 4. the use for absolute quantification
through internal standardization. The latter three applications are
quite similar, as the underlying principle is the generation of a
MS detectable mass shift compared to the unlabeled analyte.
Consequently, those applications are not restricted to deuterium
alone since a similar mass shift could also be achieved by
employing other stable isotopes (e.g.
13
C,
15
N, or
18
O). The
question whether to use deuterium or another stable isotope
label often depends on commercial availability, costs, and the
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synthetic efforts needed for label introduction. Thus, in many
cases, deuterium is preferred due to much cheaper precursor
costs and the availability of highly efficient H/D exchange
labeling approaches. However, for some specific applications
(e.g. metabolomics pathway analysis) additional considerations
may result in a preference for
13
C. Whatever the choice of
isotope, deuterium applications are typically reported in close
conjunction with other stable isotopes, which makes it almost
impossible to review deuterium applications alone.
Consequently, this section has been expanded to cover
stable isotope applications in general; however, whenever
possible, with a particular focus on deuterium.
2.1. The Kinetic Isotope Effect
[
14
]
Comparing C–H and C–D bonds, the activation energy for
the C–D bond is larger. This difference is due to the greater
mass of D versus H resulting in a lower vibrational frequency
and, thus, a lower zero-point energy (ZPE) of the C–D bond
(Scheme 2).
[
15
]
The lower energy relative to a C–H bond
translates to a higher activation energy required to reach the
transition state for bond cleavage and, thus, slower reaction rate
of deuterated analogues when the rate-determining step
involves breaking a covalent C–H/D bond.
[
16
]
This effect is
known as the primary (kinetic) hydrogen isotope effect and is
expressed as the ratio of the reaction rate constants for C–H
versus C–D bond cleavage, with expected values of k
H
/k
D
> 1
(normal KIE) or k
H
/k
D
< 1 (inverse KIE).
[
17
]
Substituting hydrogen
for deuterium, large KIEs are observed because the relative
mass change is great (100%), and even greater for tritium;
however, KIEs can also occur for other isotopes, such as
11
B,
13
C,
15
N,
18
O, and non heteroatomic bonds (e.g. D–D versus H–
H).
[14]
Scheme 2. Origins of the deuterium KIE: the lower zero-point energy (ZPE)
results in a higher activation energy for C–D bond homolysis.
Besides the semi-classical model, quantum mechanical
tunnelling and related kinetic models are also invoked to explain
KIEs.
[
18
]
The secondary hydrogen isotope effect arises in cases
where the C–H or C–D bond remains intact during the rate-
limiting step of the reaction. Secondary KIEs are typically
produced due to changes in hybridization (e.g. sp
3
to sp
2
), or the
involvement of hyperconjugation. Additionally, rates can be also
influenced by a slightly reduced steric demand of the C–D bond
(steric KIE), and the moderately increased ability of the C–D
bond to donate electron density by an inductive effect (inductive
KIE).
[
19
]
The magnitude of secondary isotope effects is much
smaller than that of the primary KIEs, and typically in the range
of ≈1.1-1.2 (normal) or ≈ 0.8-0.9 (inverse). The shifts in
equilibrium upon isotopic substitution are termed equilibrium
isotope effects (EIEs). Reactions may be also affected by the
type of solvent used (for example, changing from H
2
O to D
2
O) if
the solvent changes the isotopic composition by H/D exchange
or the solvation of the activated complex.
[
20
]
Today, KIEs can be
measured at natural abundance for nearly every type of reaction
[
21
]
or, in specific cases, even at the single-molecule level.
[
22
]
2.1.1. Isotope effects for investigation of chemical reaction
mechanism
Kinetic Isotope Effects (KIEs) have been extensively used to
study reaction mechanisms by determining rate-limiting and
product-determining steps (Scheme 3). KIEs are commonly
measured using NMR
[
23
]
to detect isotope location and/or
GC/MS and LC/MS
[
24
]
to detect mass changes. As KIEs are
typically very sensitive to substrate and transition state structure,
these measurements can be used to understand electronic,
steric, and related effects.
[
25
]
KIE experiments are often
designed and employed to support a computational hypothesis.
Thus, the change of the reaction rate following replacement of
an atom (typically hydrogen) by its isotope (deuterium) can be
compared with the theoretical KIE values to provide essential
experimental information on the calculated mechanistic
pathway.
[
26
]
Scheme 3. Complementary deuterium KIE experiments: A) KIE determined
from two parallel reactions; B) KIE determined from an intermolecular
competition; and C) KIE determined from intramolecular competition.
[27]
Three different experimental designs are typically used (see
Scheme 3). KIE determined from absolute rates of two parallel
reactions; KIE determined from an intermolecular competition
between deuterium-labeled and unlabeled substrate in the same
reaction flask; and KIE determined from an intramolecular
competition, e.g. mediated by placing a directing group (DG)
between the C–H and C–D bonds. Apart from the differences in