General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations

Article abstract of DOI:10.1002/cbic.201900740

The biocatalytic synthesis of natural and modified nucleosides with nucleoside phosphorylases offers the protecting-group-free direct glycosylation of free nucleobases in transglycosylation reactions. This contribution presents guiding principles for nucleoside phosphorylase-mediated transglycosylations alongside mathematical tools for straightforward yield optimization. We illustrate how product yields in these reactions can easily be estimated and optimized using the equilibrium constants of phosphorolysis of the nucleosides involved. Furthermore, the varying negative effects of phosphate on transglycosylation yields are demonstrated theoretically and experimentally with several examples. Practical considerations for these reactions from a synthetic perspective are presented, as well as freely available tools that serve to facilitate a reliable choice of reaction conditions to achieve maximum product yields in nucleoside transglycosylation reactions.

Full text of DOI:10.1002/cbic.201900740

Accepted Article
Title: General Principles for Yield Optimization of Nucleoside
Phosphorylase-Catalyzed Transglycosylations
Authors: Felix Kaspar, Robert T. Giessmann, Katja Hellendahl, Peter
Neubauer, Anke Wagner, and Matthias Gimpel
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To be cited as: ChemBioChem 10.1002/cbic.201900740
Link to VoR: http://dx.doi.org/10.1002/cbic.201900740
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10.1002/cbic.201900740
ChemBioChem
COMMUNICATION
General Principles for Yield Optimization of Nucleoside
Phosphorylase-Catalyzed Transglycosylations
F. Kaspar[a],#, R. T. Giessmann[b],#, K. F. Hellendahl[b], P. Neubauer[b], A. Wagner[a],[b],* and M. Gimpel[b]
[a]
[b]
#
F. Kaspar and Dr. A. Wagner
BioNukleo GmbH
Ackerstraße 76, D-13355, Berlin, Germany
E-mail: anke.wagner@tu-berlin.de
R. T. Giessmann, K. F. Hellendahl, Dr. P. Neubauer, Dr. A. Wagner and Dr. M. Gimpel
Department of Biotechnology
Technical University of Berlin
Ackerstraße 76, ACK24, D-13355, Germany
the authors contributed equally
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: The biocatalytic synthesis of natural and modified
nucleosides with nucleoside phosphorylases offers the protecting
group-free direct glycosylation of free nucleobases in
transglycosylation reactions. This contribution presents guiding
principles for nucleoside phosphorylase-mediated transglycosylations
alongside mathematical tools for straightforward yield optimization.
We illustrate how product yields in these reactions can easily be
estimated and optimized using the equilibrium constants of
phosphorolysis of the nucleosides involved. Furthermore, the varying
negative effects of phosphate on transglycosylation yields are
demonstrated theoretically and experimentally with several examples.
spectrum, excellent tolerance to harsh reaction conditions as well
as perfect regio- and diastereoselectivity at the C1’ position.[11,12]
Practical considerations for these reactions from
a synthetic
perspective are presented, as well as freely available tools that serve
to facilitate a reliable choice of reaction conditions to achieve maximal
product yields in nucleoside transglycosylation reactions.
Scheme 1. Reaction sequence of a nucleoside transglycosylation.
Nucleosides are highly functionalized biomolecules essential for
the storage of information as DNA and RNA, cellular energy
transfer and as enzyme cofactors. Modified nucleosides are
widely employed as pharmaceuticals for the treatment of cancers
and viral infections.[1] Consequently, their synthetic accessibility is
crucial. However, the preparation of nucleosides and nucleoside
analogs by conventional synthetic methods heavily relies on
protecting groups and, thus, suffers from poor atomic efficiency
and low yields.[2–5]
Biocatalytic methods offer the efficient and protecting group-free
synthesis of pyrimidine and purine nucleosides. The use of
nucleoside phosphorylases (NPases) for the preparation of
nucleosides and their analogues in transglycosylation reactions is
firmly established[6] and numerous examples of enzymatic or
chemoenzymatic syntheses can be found in the literature.[7–14]
NPases catalyze the reversible phosphorolysis of nucleosides to
pentose-1-phosphates (Scheme 1, I). In transglycosylation
reactions, a forward and a reverse nucleoside phosphorolysis are
coupled in situ to glycosylate a free nucleobase with the pentose-
1-phosphate generated by the first reaction (Scheme 1, I and II).
Formally, this equals a direct glycosylation of the nucleobase to
yield a nucleoside of interest. Conveniently, nature has provided
an arsenal of robust biocatalysts that offer a broad substrate
Despite their great versatility, enzymatically catalyzed nucleoside
transglycosylation reactions have previously suffered from an
unclear interrelation between yields and the employed enzymes
and starting materials. Particularly, the impact of different sugar
donors and/or nucleobases as well as varying phosphate
concentrations on the product yield had remained unclear until
recently. The pioneering work of Alexeev et al.[15] demonstrated
that yields of nucleoside transglycosylation reactions involving
uridine and adenosine can be accurately predicted based on the
equilibrium constants of phosphorolysis of the sugar donor and
the product nucleoside. They concluded that the ratio of the
equilibrium constants of the sugar donor and the product
nucleoside (퐾1/퐾2) determines maximum product yields and that
an excess of sugar donor is further beneficial. On the other hand,
increasing phosphate concentrations were shown to have a
negative impact on product yields. However, Alexeev and
colleagues[15] based their calculations on the assumption that the
concentration of phosphate is constant and furthermore only
investigated one example of a high-yielding NPase-catalyzed
transglycosylation. As a continuation of the considerations of
Alexeev et al.[15] we explored this reaction system from a practical
1
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Figure 1. Impact of different 퐾1 and
퐾2 values on transglycosylation
yield and phosphate gap. Realistic
퐾1 and 퐾2 values were assumed
based
equilibrium
on
recently
reported
The
constants.[17]
graphs for maximal yield (max.;
black), 0.1 eq. (green), 1 eq. (blue)
and 10 eq. (red) of phosphate were
plotted using numerical solutions of
the
system
of
equilibrium
constraints (1) and (2) calculated
with the Python code described in
the
external
supplementary
material.[16]
[
]
퐵2 [푃1푃]
synthetic perspective and developed a universally applicable
equation for yield prediction. We show that the yield-diminishing
effect of phosphate strongly and mainly depends on the
equilibrium constant of phosphorolysis of the nucleoside of
interest (퐾2). This important feature, which proved critical in the
biocatalytic preparation of pharmaceutically relevant pyrimidine
nucleosides has thus far not been described theoretically or
experimentally. Alongside our freely available Python code for
precise yield predictions (see below) we also provide a simplified
equation for the estimation of product yield that allows for
straightforward analytical solutions instead of the numerical
solutions previously required.
Nucleoside transglycosylation reactions are generally considered
as formal glycosylations of a nucleobase B2, which yields the
corresponding nucleoside of interest, N2. Here, a starting
nucleoside, N1, is used as a glycosylation agent with the purpose
of donating the sugar moiety. In an enzyme cascade, the sugar
donor N1 is subjected to phosphorolysis yielding a pentose-1-
phosphate P1P, which is consumed in the sequential reaction with
nucleobase B2 to produce N2 (Scheme 1). The yield of this
reaction is generally defined as the formation of N2 in respect to
B2, neglecting the other reagents P1P, B1, N1 and phosphate.
Indeed, inorganic phosphate only plays a catalytic role as it is
used in the first step but liberated again in the following reaction.
Generally, yields in NPase-catalyzed transglycosylations are
dictated by the equilibrium constraints of the two half reactions I
and II:
퐾2 =
(2)
[
]
푁2 [푃]
where 퐾1 and 퐾2 are the apparent equilibrium constants of
phosphorolysis of the sugar donor and product nucleoside,
respectively, [푃] is the equilibrium concentration of phosphate,
[푃1푃] is the equilibrium concentration of the pentose-1-phosphate
[
] [ ] [
]
[
]
and 푁1 , 푁2 , 퐵1 and 퐵2 are the equilibrium concentrations
of the nucleosides and bases. Alexeev et al.[15] previously solved
this system of equations by assuming a constant concentration of
phosphate and numerically solving the resulting cubic equation.
When we attempted to apply their equations to the synthesis of
the pharmaceutically relevant nucleoside 5-ethynyluridine we
were unable to obtain results that were in agreement with
experimental HPLC data, as their formula yielded negative values
for this case (see Supplementary Table S1). Therefore, we sought
establish a mathematical tool that allows general applicability and
reaction optimization of all nucleoside transglycosylations.
Bypassing the simplification made by Alexeev and coworkers, we
implemented the system of equilibrium constrains (1) and (2)
including all reagents as variables in a Python code to obtain more
precise predictions (see externally hosted Python code).[16]
Numerical solutions of this system allowed theoretical
examination of the effect of phosphate and sugar donor excess
on the product yield, considering a reasonable range of
equilibrium constants.[17] Approaching zero phosphate
concentration, the maximum (ideal) product yield can be obtained,
but at higher phosphate concentrations an apparent loss of yield
can be observed due to phosphorolysis (or non-synthesis) of the
product nucleoside (Figure 1). While the 퐾1/퐾2 ratio (equal to 퐾푁)
dictates the maximal yield with minimal phosphate, 퐾2 determines
the extent of yield loss in the presence of phosphate. A high 퐾푁 in
the order of 5—15 promises good to excellent yields (i.e. > 90%)
[
]
퐵1 [푃1푃]
퐾1 =
(1)
[
]
푁1 [푃]
2
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10.1002/cbic.201900740
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COMMUNICATION
Figure 2. Biocatalytic synthesis of nucleosides by transglycosylation. Reactions were performed with 1 mM uridine as sugar donor (퐾1
= 0.16), 0.5 mM nucleobase, 32 µg mL-1 pyrimidine NPase (2.5 U mL-1) and 66 µg mL-1 purine NPase (5.0 U mL-1) in 50 mM glycine
buffer at pH 9 and 60 °C with either 0.1 mM (0.2 equivalents in respect to the starting base), 0.5 mM (1 eq.) or 5 mM (10 eq.) K2HPO4
in a total volume of 1 mL. Experimental yield (empty diamonds) was determined by HPLC considering conversion of the free nucleobase
to its corresponding ribosyl nucleoside. Predictions (blue, light blue, turquoise and green columns) were carried out with the Python
code described in the external supplementary material.[16] The values for the maximal yield (max.; blue) can also be obtained via
equation (4).
with only moderate excesses (i.e. 2—fold) of the sugar donor. On
the other hand, reactions with a low 퐾푁 require a great excess of
sugar donor to facilitate yields upwards of 50%. Interestingly, the
effect of phosphate varies between systems with the same 퐾푁,
which results from the fact that high 퐾2 values dictate a greater
degree of phosphorolysis of N2 at non-negligible phosphate
concentrations – even at great excess of the sugar donor N1
(Figure 1). Notably, while potential formation of intermediate
pentose-1-phosphate needs to be considered for a realistic
assessment and prediction of synthetic yield, we only observed
less than 4 percentage points of deviation from the ideal yield for
any nucleoside transglycosylation with < 0.3 eq. of phosphate in
the reaction conditions we covered with our considerations.[16]
To validate these predictions experimentally and demonstrate the
varying impact of phosphate on the product yield, we prepared a
series of natural and base-modified ribosyl nucleosides from their
respective nucleobases, using uridine as a sugar donor. Fitting of
the experimental data to the equilibrium constraints[15,16] yielded
equilibrium constants 퐾1 and 퐾2 very similar to those reported
previously[17] and revealed a great range of apparent equilibrium
constants 퐾2 (0.01 to 0.35 at 60°C, pH 9) and 퐾푁 (0.4 to 16.0). In
all cases, the experimental yields determined by HPLC agreed
well with the predictions obtained for different phosphate
concentrations (Figure 2). Our data emphasize that, as illustrated
in Figure 1, particularly the yields of transglycosylation reactions
with high 퐾2 values suffer enormously from phosphate
concentrations higher than strictly necessary. For instance,
adenosine formation (퐾2 = 0.02) was impacted only minorly by the
addition of 10 eq. of phosphate (92% ideal yield vs. 88%
experimental yield with 10 eq. of phosphate), but 5-ethynyluridine
yield (퐾2 = 0.35) dropped by more than 30 percentage points
under the same conditions (53% ideal yield vs. 22% experimental
yield with 10 eq. of phosphate; Figure 2). Thus, steep losses in
yield should be expected for products with a high 퐾 value (퐾2),
whereas the synthesis of nucleosides with low 퐾 values tolerates
significant amounts of phosphate (Figure 2). Consistent with our
predictions, we only observed small deviations from the maximal
yield in the experiments with 0.2 equivalents of phosphate. Thus,
the concentration of phosphate should be kept low in synthetic
nucleoside transglycosylations to obtain maximal yield. For these
cases, calculation of maximal (ideal) conversion provides a close
approximation of the yield and allows for the use of a simplified
formula.
Considering ideal (intermediate-free) coupling of the two half
reactions, I and II, the terms for phosphate and P1P would cancel
in the mathematical consideration of this system, as the
production of these in one half reaction is compensated by the
consumption in the other. Considering the net reaction, one may
therefore define:
[
]
퐾1
푁2 [퐵1]
퐾푁
=
=
(3)
[
]
퐾2
푁1 [퐵2]
with definitions from above. Solving this equation for the
concentration of the product nucleoside, N2, yields only one
physically possible solution that can be used to calculate ideal
(phosphate-
and
P1P-free)
yields
of
nucleoside
transglycosylations with variable initial concentrations of the
[
]
[
]
sugar donor N1 and the nucleobase B2, 푁1 and 퐵2
,
0
0
respectively:
([
]
[
] )
퐵2
0
퐾푁 푁1
+
0
[
]
푁2
=
−
(
)
2 퐾푁 − 1
(4)
2
퐾푁 퐾푁 푁1 2 − 2퐾푁 푁1 퐵2 0 + 퐾푁 퐵2 + 4 푁1 퐵2
(
√
[
]
[
] [
]
[
]
[
] [ ] )
0
0
0
0
0
2(퐾푁 − 1)
Thus, the maximal yield (at zero phosphate) can be calculated
easily via equation (4) to reflect a realistic estimate of the
experimental yield if < 0.3 eq. of phosphate are used. Ideal yields
for conversions employing a range of pyrimidine and purine
3
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COMMUNICATION
ribosyl and 2’-deoxyribosyl nucleosides[17] with different reaction
conditions including sugar donor excess and temperature, can be
calculated with an Excel sheet freely available from the externally
hosted supplementary material.[18]
Einstein Center of Catalysis (EC2). K.H. is funded by the Deutsche
Forschungsgemeinschaft (DFG), grant number 392246628.
These considerations and previous findings[15,16] bear several
Keywords: nucleoside • nucleoside phosphorylase • phosphate
• pentose-1-phosphate • equilibrium constant
practical
implications
for
NPase-catalyzed
nucleoside
transglycosylations. First, a high 퐾1 /퐾2 ratio (high 퐾푁 ) leads to
excellent yields which can be achieved with moderate excess of
the sugar donor, as mentioned by Alexeev and colleagues,[15] and
estimated easily with equation (4). Second, pyrimidine
nucleosides serve better as sugar donors than purine
nucleosides.[17] From a practical point of view, uridine and
thymidine recommend themselves as ribosyl and 2’-deoxyribosyl
donor, respectively, due to their simple commercial availability
and high 퐾 value. Third, phosphate concentration in the
transglycosylation reaction should generally be kept as low as
possible to prevent loss of product yield. This becomes especially
important in the synthesis of nucleosides with high 퐾 values, such
as pyrimidine nucleosides like 5-ethynyluridine. Thus, 0.1—0.3
equivalents of phosphate in respect to the starting base may
present an appropriate trade-off between reaction speed and
maximal yield. A potential workflow for the fruitful application of
the methodology presented in this work is suggested in the
supporting information.
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Given the easy accessibility of apparent equilibrium constants of
phosphorolysis of any nucleoside of interest, the tools for yield
prediction presented in this work aid the straightforward design
and optimization of nucleoside transglycosylations to facilitate
high yields in NPase-catalyzed reactions. Exact yield prediction of
transglycosylations may be performed with our Phyton code
considering phosphate[16] and practical estimations for ideal yield
can easily be obtained via equation (4).[18]
[13] J. Wierzchowski, A. Stachelska-Wierzchowska, B. Wielgus-Kutrowska,
A. Bzowska, Curr. Pharm. Des. 2017, 23, 6948–6966.
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Experimental Section
[16] R. T. Giessmann, 2019, 10.5281/zenodo.3522588.
[17] F. Kaspar, R. T. Giessmann, P. Neubauer, A. Wagner, M. Gimpel, Adv.
Synth. Catal. 2019, in press.
Enzymatic nucleoside transglycosylations were performed with
0.5 mM nucleobase, 1 mM uridine as sugar donor, 32 µg mL-1
pyrimidine NPase (2.5 U mL-1; E-PyNP-0002, BioNukleo GmbH,
Berlin, Germany) and 66 µg mL-1 purine NPase (5.0 U mL-1; E-
PNP-0002, BioNukleo GmbH) in 50 mM glycine buffer at pH 9 and
60 °C with either 0.1 mM (0.2 equivalents in respect to the starting
base), 0.5 mM (1 eq.) or 5 mM (10 eq.) K2HPO4 in a total volume
of 1 mL. Reaction mixtures were prepared from stock solutions
and started by the addition of the enzyme(s). Time to equilibrium
was approximated via UV/Vis spectroscopy.[19] Allowing for
additional time after apparent reaction completion, the reactions
were stopped after 1 h by quenching samples of 100 µL in an
equal volume of MeOH and analyzed by HPLC. All experimental
and calculated data are available online.[16,18]
[18] F. Kaspar, R. T. Giessmann, 2019, 10.5281/zenodo.3565561.
[19] F. Kaspar, R. T. Giessmann, N. Krausch, P. Neubauer, A. Wagner, M.
Gimpel, Methods Protoc. 2019, 2, 60.
Acknowledgements
We would like to thank Sarah von Westarp for initial support.
Funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) under Germany´s Excellence
Strategy – EXC 2008/1 – 390540038. We are grateful for the
support of R.T.G. by the Einstein Foundation Berlin (ESB) -
4
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Entry for the Table of Contents
Not all nucleosides are created equal: The equilibrium constants of phosphorolysis of different nucleosides vary drastically, and so
does their yield and behavior in biocatalytic transglycosylation reactions. The data, mathematical tools and guidelines presented here
facilitate the reliable choice of reaction conditions for maximum yield in these reactions.
5
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