Thermodynamic Reaction Control of Nucleoside Phosphorolysis

Article abstract of DOI:10.1002/adsc.201901230

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis. (Figure presented.).

Full text of DOI:10.1002/adsc.201901230

Title: Thermodynamic Reaction Control of Nucleoside Phosphorolysis
Authors: Felix Kaspar, Robert Giessmann, Peter Neubauer, Anke
Wagner, and Matthias Gimpel
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201901230
Link to VoR: http://dx.doi.org/10.1002/adsc.201901230

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201901230
Thermodynamic Reaction Control of Nucleoside Phosphorolysis
Felix Kaspara,b,#, Robert T. Giessmanna,#, Peter Neubauera, Anke Wagnera,b,* and
Matthias Gimpela
a
Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstraße 76, ACK24, D-
13355 Berlin, Germany
anke.wagner@tu-berlin.de, Tel.: +49 30 314 72183
BioNukleo GmbH, Ackerstraße 76, D-13355 Berlin, Germany
b
# the authors contributed equally
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. Nucleoside analogs represent a class of important the apparent transformed reaction enthalpy and apparent
drugs for cancer and antiviral treatments. Nucleoside transformed reaction entropy for 24 nucleosides, confirming
phosphorylases (NPases) catalyze the phosphorolysis of that these conversions are thermodynamically controlled
nucleosides and are widely employed for the synthesis of endothermic reactions. This data allows calculation of the
pentose-1-phosphates and nucleoside analogs, which are Gibbs free energy and, consequently, the equilibrium
difficult to access via conventional synthetic methods. constant of phosphorolysis at any given reaction
However, for the vast majority of nucleosides, it has been temperature. Overall, our investigations revealed that
observed that either no or incomplete conversion of the pyrimidine nucleosides are generally more susceptible to
starting materials is achieved in NPase-catalyzed reactions. phosphorolysis than purine nucleosides. The data disclosed
For some substrates, it has been shown that these reactions are in this work allow the accurate prediction of phosphorolysis
reversible equilibrium reactions that adhere to the law of mass or transglycosylation yields for a range of pyrimidine and
action. In this contribution, we broadly demonstrate that purine nucleosides and thus serve to empower further
nucleoside phosphorolysis is a thermodynamically controlled research in the field of nucleoside biocatalysis.
endothermic reaction that proceeds to a reaction equilibrium
dictated by the substrate-specific equilibrium constant of
phosphorolysis, irrespective of the type or amount of NPase
used, as shown by several examples. Furthermore, we
explored the temperature-dependency of nucleoside
phosphorolysis equilibrium states and provide
Keywords: nucleosides; nucleoside phosphorylase;
nucleoside phosphorolysis; equilibrium constant;
temperature
phosphorylases (Py-NPases) or purine nucleoside
phosphorylases (Pu-NPases), depending on their
substrate spectra.[4]
Introduction
Nucleosides serve as drugs against a variety of cancers
and viral infections.[1] Thus, their cost- and time-
efficient preparation is of high interest. However, the
synthesis of nucleosides and nucleoside analogs via
conventional synthetic methods comes with several
challenges posed by regio- and stereochemical
complexity, functional group sensitivity and,
consequently, heavy reliance on protecting groups.[2]
Biocatalytic methods are a valuable alternative as
pyrimidine and purine nucleosides can be synthesized
with high selectivity in sustainable enzyme-catalyzed
processes.[3] Here, the use of nucleoside
phosphorylases (NPases) has been firmly established
and these enzymes are widely applied for the synthesis
of nucleosides and their analogues.[3]
Scheme
1.
Generalized
biocatalytic
nucleoside
phosphorolysis reaction. A nucleoside is subjected to
phosphorolytic cleavage of the nucleobase, yielding a
pentose-1-phosphate and a free nucleobase.
The use of NPases as biocatalysts in organic
chemistry offers the advantage of using mild aqueous
reaction conditions, a broad substrate spectrum
regarding sugar and base moieties, as well as perfect
regio- and stereoselectivity at the C1’ position.
Employing these enzymes, pentose-1-phosphates can
be obtained as important synthetic intermediates from
NPases catalyze the phosphorolysis of nucleosides
to pentose-1-phosphates, as well as the corresponding
reverse reaction (Scheme 1). They are generally
classified
as
either
pyrimidine
nucleoside
1
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
the accessible pool of nucleosides[5,6] which have been
shown to be highly valuable precursors for the
synthesis of base- and sugar-modified nucleosides.[7–9]
Furthermore, NPases are widely used for the synthesis
of nucleoside analogues in transglycosylation
reactions.[10–12]
Current research activities mainly focus on kinetic
aspects of individual NPase-catalyzed reactions.
Hence, kinetic data for a variety of NPases are
nucleoside phosphorolysis proceeds to a reaction
equilibrium dictated by the substrate-specific
equilibrium constant of phosphorolysis, irrespective of
the type or amount of NPase used. Furthermore, we
show that the equilibrium constant is temperature
dependent. Therefore, we determined the equilibrium
constants of phosphorolysis of 24 nucleosides at
different temperatures and derived the apparent
transformed enthalpies and entropies. The resulting
available. To increase the final yield of phosphorolysis, data show that pyrimidine nucleosides are generally
mainly the choice of enzyme,[13,14] enzyme
immobilization[15] or enzyme engineering[16,17] have
been investigated. Nonetheless, NPases generally fail
to facilitate full conversion.[18] A recent report by
Alexeev and coworkers has shed more light on the
cause of this phenomenon and showed that the
thermodynamic properties of the nucleosides involved
in the reaction influence the yields of NPase-catalyzed
reactions.[19]
Since nucleoside phosphorolysis is a reversible
reaction (Scheme 1), there is a point, where the rates
of the forward and the backward reactions are equal
and no apparent change in concentrations is observed.
In this equilibrium state, the reaction quotient defined
as the quotient of the concentrations of the products
and the substrates can be derived from the law of mass
action and is generally referred to as the apparent
equilibrium constant 퐾′.[20] The following expression
results for nucleoside phosphorolysis reactions:
more susceptible to phosphorolysis than purine
nucleosides. Additionally, our enthalpy and entropy
data allow calculation of equilibrium constants at
different temperatures as well as prediction of
phosphorolysis yields, as demonstrated herein.
Results and Discussion
Maximum conversions of nucleosides in
phosphorolysis reactions are independent of the
applied biocatalyst
Previous research activities have focused on the
discovery of new enzymes[13,14] or enzyme
immobilization strategies[15] to increase the obtained
yield
for
nucleoside
phosphorolysis
and
transglycosylation reactions. Based on the
thermodynamic characteristics of these reactions,
however, we anticipated that the maximum conversion
in phosphorolysis reactions would behave
independently of the enzyme used.
[ ]
퐵 [푃1푃]
퐾′ =
(1)
To investigate the impact of different NPases on
the equilibrium states of nucleoside phosphorolysis
reactions, we initially performed the phosphorolysis of
uridine (1) with Escherichia coli uridine
phosphorylase (E. coli UP), E. coli thymidine
phosphorylase (E. coli TP), Bacillus subtilis Py-NPase
(B. subtilis Py-NPase) and two commercially available
thermostable Py-NPases (Py-NPase Y01 and Py-
NPase Y02). All enzymes readily accepted this
substrate and reaction completion could be observed
after 5 to 80 min using 10 µg∙ml-1 of the respective
enzymes (Figure 1A). Using 5 equivalents (eq.) of
phosphate with respect to the nucleoside substrate, a
maximum conversion of 55% was achieved regardless
of the enzyme used.
To exclude any concentration effects of the
enzyme preparations, we additionally conducted the
phosphorolysis of 1 with different concentrations of
Py-NPase Y02 (Figure 1B). Higher enzyme
concentrations led to faster reaction completion.
However, irrespective of the amount of the enzyme
used, the same equilibrium as previously observed was
reached.
[ ]
푁 [푃]
where 퐾′ is the apparent equilibrium constant of
[ ]
phosphorolysis, 퐵 is the equilibrium concentration
of the free nucleobase [mM], [푃1푃] is the equilibrium
[ ]
concentration of the pentose-1-phosphate [mM], 푁
is the equilibrium concentration of the nucleoside
[mM] and [푃] is the equilibrium concentration of
inorganic phosphate [mM].
This equilibrium constant exists irrespective of the
(bio)catalyst used and 퐾′ values for some substrates in
NPase-catalyzed reactions have been reported.[19,21–26]
Thus, according to the law of mass action, only the
reaction conditions influence the final equilibrium
concentrations and not the enzyme used for catalysis.
Alexeev and colleagues[19] recently employed this
dependency in their method for accurate yield
prediction of nucleoside transglycosylation reactions
based on the equilibrium constants of the individual
reactions. Ultimately, this allows for thermodynamic
reaction control by adjusting the concentrations of the
starting materials to facilitate an optimal yield.
In this work we provide extensive evidence for the
universal interpretation of nucleoside phosphorolysis
To eliminate the possibility of enzyme
inactivation preventing the completion of the reaction,
we performed the phosphorolysis of 1 with Py-NPase
Y02 and added additional enzyme after apparent
reaction completion (Figure 1C).
reactions
as
thermodynamically
controlled
endothermic equilibrium reactions that adhere to the
law of mass action. To this end, we performed and
monitored
several
biocatalytic
nucleoside
phosphorolysis reactions. We demonstrate that
2
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
Figure 1. Enzymatic phosphorolysis of uridine (1). A Phosphorolysis of 1 with five different Py-NPases. B Phosphorolysis
of 1 with Py-NPase Y02 using different enzyme concentrations. C Phosphorolysis of 1 with Py-NPase Y02 employing
spiking of enzyme upon apparent reaction completion. All reactions reach the same equilibrium, regardless of the enzyme
or its amount used for catalysis. Reactions were performed with 2 mM nucleoside substrate and 10 mM K2HPO4 in 50 mM
MOPS buffer at pH 7.5 and 37 °C in a total volume of 500 µL. 10 µg∙ml-1 of the respective enzyme were used in A and 10—
40 µg∙ml-1 Py-NPase Y02 were used in B. The reaction in C was started with 40 µg∙ml-1 (20 µg total enzyme) Py-NPase Y02
and 10 µg of the enzyme were added at 10 min and 15 min each, indicated by the arrows. Reactions in A were performed in
duplicate and the standard deviation (SD) is shown as error bars.
This experiment resulted in no change of reactant
concentrations after addition of more enzyme,
confirming that this reaction was not terminated by
means of inhibition and/or enzyme inactivation.
Interestingly, Py-NPases Y01, Y02 and B. subtilis Py-
NPase performed roughly equally well with uridine (1)
compared to 2’-deoxyuridine (2), whereas our data
nicely reflect the inverse substrate specificity of E. coli
UP and TP, as reported previously.[29,30] E. coli UP
showed excellent activity with uridine (1) but
significantly diminished activity with the 2’-deoxy
analogue 2. E. coli TP, on the other hand, displayed
only low activity with 1 but facilitated remarkably
quick reaction completion with 2 (Figure 1A and 2A).
Notably, although it is commonly believed that
Pu-NPases should be specific to purine nucleosides,
they generally also catalyze the phosphorolysis of
pyrimidine nucleosides. Compared to corresponding
Py-NPases they usually exhibit >2,000-fold lower
turnover rates. Nonetheless, given the thermodynamic
reaction control of nucleoside phosphorolysis, Pu-
NPases should still be expected to complete the
phosphorolysis of their unfavored substrates, despite
their kinetic handicap. Consequently, for the
phosphorolysis of the pyrimidine 5-fluorouridine (3),
This evidence confirms that the phosphorolysis of
uridine (1) is a thermodynamically controlled
equilibrium reaction which is consistent with previous
reports.[19,21–24,27] From the data collected, the apparent
equilibrium constant of phosphorolysis 퐾′ under these
conditions was 0.15, as calculated from equation (1).
To broadly confirm the thermodynamic reaction
control of nucleoside phosphorolysis, we investigated
the conversion of other nucleosides by NPases. We
observed a similar behavior for the phosphorolysis of
the pyrimidine nucleosides 2’-deoxyuridine (2), 5-
fluorouridine (3) and 2’-deoxy-5-fluorouridine (4),
employing the same Py-NPases (Figure 2A—2C). As
observed for uridine (1), the choice of enzyme had no
effect on the equilibrium conversions. Pyrimidines
2—4 displayed very similar apparent equilibrium
constants to 1 (0.20 for 2, 0.14 for 3 and 4).
To extend this investigation to purine nucleosides, even the use of the thermostable Pu-NPase N02 proved
we performed the phosphorolysis of two natural
(adenosine, 5, and 2’-deoxyadenosine, 6) and two
modified purine nucleosides (2-chloroadenosine, 7,
and 2-chloro-2’-deoxyadenosine 8) with three
different NPases (thermostable Pu-NPases N01 and
N02 and E. coli Pu-NPase). Similar to the pyrimidine
nucleosides mentioned above, the choice of the
enzyme had no effect on the equilibrium
concentrations of these reactions (Figure 2D—2G).
Here, the equilibrium conversions with 5 eq. of
phosphate were significantly lower, in the range of
20%. Calculation of the apparent equilibrium
constants of phosphorolysis yielded values around
0.01, which is consistent with previous reports for
5.[19,28]
successful in reaching the thermodynamic equilibrium
(Figure 2B). In this case, a 60-fold higher amount of
enzyme had to be employed and the reaction took
considerably longer than with any of the Py-NPases
under the same reaction conditions. Thus far, to the
best of our knowledge, only the phylogenetically
peculiar Plasmodium falciparum Pu-NPase has been
reported to perform the phosphorolysis of a pyrimidine
nucleoside.[31] Here, we not only show for the first time
that the thermostable Pu-NPase N02 accepts
pyrimidine substrates such as 3, but also achieves
reaction completion given sufficient time. This
strikingly emphasizes the non-significance of the
enzyme kinetics for the ultimate reaction outcome, as
3
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
Figure 2. Enzymatic phosphorolysis of pyrimidine and purine nucleosides. The phosphorolysis of A 2’-deoxyuridine (2), B
5-fluorouridine (3), C 2’-deoxy-5-fluorouridine (4), D adenosine (5), E 2’-deoxyadenosine (6), F 2-chloroadenosine (7) and
G 2-chloro-2’-deoxyadenosine (8) was performed in duplicate with 2 mM nucleoside substrate, 10 mM K2HPO4 and
10 µg∙ml-1 of the respective enzyme (except for the phosphorolysis of 3 with Pu-NPase N02, where 600 µg∙ml-1 were used)
in 50 mM MOPS buffer at pH 7.5 and 37 °C in a total volume of 500 µL. Note the different time scales. Error bars show the
SD.
it is possible to drive these phosphorolysis reactions
into their chemical equilibrium even with an extremely
small enzyme activity, in a range commonly
considered negligible.
nucleosides that are not converted by NPases because
of issues such as excessive steric demands or severely
unfavored
transition
states
which
prevent
transformation.
Thus, all nucleoside phosphorolysis reactions we
investigated behaved according to the law of mass
action and reached maximum product concentrations
after a variable run time. Despite differences in
reaction speed, the maximum conversion yields in the
equilibrium are dictated by a characteristic and
substrate-specific equilibrium constant which was
shown to be independent of the type and amount of
enzyme used. It may be reasonable to assume that this
holds true for all nucleosides that are subjected to
phosphorolysis. Important exceptions from this are
Higher reaction temperature yields higher
conversion of nucleosides by NPases
The absolute values of the equilibrium constants of
phosphorolysis of the nucleosides discussed above
were all found to be well below 1. This means that they
describe endothermic reactions, which are not favored
under standard conditions. Consequently, we
anticipated a temperature-dependence of 퐾′ and set
out to investigate the effect of higher reaction
temperatures on the equilibrium states of nucleoside
4
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
phosphorolysis. Profiting from the use of the
thermostable NPases Y02 and N02, we were able to
perform the phosphorolysis of 24 nucleosides,
including 12 pyrimidine and 12 purine nucleosides, at
temperatures of 40 to 70 °C. To prevent decomposition
of the produced pentose-1-phosphates, which is
known to happen rapidly at high temperatures and
neutral pH values,[5] we increased the reaction pH to 9.
Under these conditions, both enzymes displayed
excellent activity with all substrates 1—24 and
allowed efficient reaction completion and monitoring.
Consistent with our initial assumptions, we observed a
trend towards higher conversions at higher
temperatures. For example, under these conditions the
phosphorolysis of 2’-deoxy-5-fluorouridine (4; Figure
3A) showed equilibrium conversions in the range of
51% at 40 °C and values around 55% at 70 °C. A
similar behavior could be observed for all nucleosides
investigated which confirms that the phosphorolysis of
1—24 are thermodynamically controlled endothermic
reactions (Table S1).
phosphorolysis ∆푅퐺′ at 40 °C of pyrimidine
nucleosides was found to be in the range of 1.2—
5.5 kJ∙mol-1, which is notably lower than the
investigated purine nucleosides that display values of
5.7—12.6 kJ∙mol-1 (Figure 4A). These values
correspond to apparent equilibrium constants 퐾′ in the
range of 0.12—0.62 for pyrimidine nucleosides and
0.01—0.11 for purine nucleosides. Accordingly,
noticeably different equilibrium conversions can be
observed across these nucleosides, with pyrimidines
generally featuring higher equilibrium conversions
than purines, as dictated by their respective
equilibrium constants (Figure 4B).
The difference between the equilibrium
conversions determined at pH 9 (this section) and the
ones determined at pH 7.5 (see above) were
insignificant (compare Figure 1 and Figure 2 with
Table S1). Although a difference between them might
be expected from the definition of the biochemical
equilibrium, it seems that pH only minorly influences
the equilibrium constant by changing the contribution
of charged species.
Pyrimidine nucleosides are generally more
susceptible to phosphorolysis than purine
nucleosides
From the described data, we derived 퐾′ at various
temperatures and determined the transformed apparent
reaction enthalpy ∆푅퐻′∗ and transformed apparent
reaction entropy ∆푅푆′∗ via fitting of the data to:
′
′∗
′∗
퐾′ = 푒− ∆ 퐺 = 푒− ∆ 퐻 − 푇∆ 푆
푅
푅푇
푅
푅
Figure 3. Examples of nucleoside phosphorolysis
thermodynamic data processing. A Raw data of the
phosphorolysis of 4 at different temperatures. The reactions
were performed with 2 mM nucleoside substrate, 10 mM
K2HPO4 and 16 µg∙ml-1 Py-NPase Y02 in 50 mM glycine
buffer at pH 9 and 40—70 °C in a total volume of 500 µL.
B Transformed and fitted data for the derivation of ∆푅퐻′∗
and ∆푅푆′∗. Raw data were processed via equation (1) and
then fitted with equation (2).
(2)
푅푇
where ∆푅퐺′ is the transformed apparent Gibb’s free
energy of phosphorolysis [J∙mol-1], 푇 is the
temperature [K], 푅 is the universal gas constant (8.314
J∙mol-1∙K-1), ∆푅퐻′∗ is the transformed apparent
enthalpy of phosphorolysis [J∙mol-1], ∆푅푆′∗ is the
transformed apparent entropy of phosphorolysis
[J∙mol-1∙K-1], and the definitions from above.
Here, we carried out direct determinations
through a non-linear fit to prevent error propagation by
linearization (Figure 3A and 3B; see Table S1 for all
derived values for ∆푅퐻′∗ and ∆푅푆′∗ ; note that we
communicate transformed thermodynamic values, as
they were measured at constant pH and do not consider
charged species, and explicitly abstain from referring
to our results as “standard” values, but apparent ones,
as they were not measured under standard biochemical
conditions).
The practical value of our collection of ∆푅퐻′∗ and
∆푅푆′∗ data lies within their use for the prediction of the
equilibrium phosphorolysis conversion of the
nucleosides investigated here. Using equation (2), the
equilibrium constants of phosphorolysis of any of the
nucleosides investigated here can easily be obtained
for any given temperature. Employing then:
[ ]
[
]
퐵 = 푃1푃 =
′([ ]
푁
[ ] )
0 + 푃
퐾
0
−
The knowledge of ∆푅퐻′∗ and ∆푅푆′∗ then allowed
access to the transformed apparent Gibb’s free energy
∆푅퐺′ of these phosphorolysis reactions at different
temperatures. The Gibb’s free energy of
2 퐾 − 1
′
(
)
(3)
2
′
′[ ]
′[ ]
(
)
′[ ] [ ]
√(퐾 푁 0 + 퐾 푃 ) − 4 퐾 − 1 퐾 푁
푃
0
0
0
2(퐾′ − 1)
5
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
Figure 4. Gibb’s free energy of phosphorolysis and conversion of nucleosides. A Gibb’s free energy of phosphorolysis
determined via equations (1) and (2) from the equilibrium concentrations of nucleoside phosphorolysis reactions with 2 mM
nucleoside substrate, 10 mM K2HPO4 and 16 µg∙ml-1 Py-NPase Y02 or 66 µg∙ml-1 Pu-NPase N02 in 50 mM glycine buffer
at pH 9 in a total volume of 500 µL performed at 40—70 °C. Error bars quantify the uncertainty of the prediction at 25 °C
calculated via equation (4). B Equilibrium conversion of nucleosides in a phosphorolysis reaction at 40 °C and pH 9
employing 5 eq. of phosphate and equilibrium constant 퐾′ (also see Table S1). [a] Rib = ribosyl, dRib = 2’-deoxyribosyl,
[b] extrapolated from experimental data for ∆푅퐻′∗ and ∆푅푆′∗, [c] experimental data (conditions as above), [d] calculated
from data for 50—70 °C since data for 40 °C were excluded from analysis (see Supplementary Material for full dataset).
[ ]
where
푁
is the initial concentration of the
pH 9 and 40 °C with different concentrations of
phosphate (Figure 5). For example, pyrimidine 1
reached a conversion of 80% with 20 eq. of phosphate
whereas for purine 5 we only observed a conversion of
37% under the same conditions, which is consistent
with the predictions obtained via our collection of
∆푅퐻′∗ and ∆푅푆′∗ data (Table S1). This emphasizes the
practical value of the knowledge of the equilibrium
constants of phosphorolysis of these nucleosides, as
they allow for accurate prediction of phosphorolysis
yields to optimize the reaction conditions.
0
[ ]
nucleoside [mM], 푃 is the initial concentration of
0
phosphate [mM] and definitions from above yields the
concentrations of the free nucleobase and pentose-1-
phosphate with variable initial concentrations of
nucleoside and phosphate. Thus, conversions of these
nucleosides can be predicted. To ease these
calculations, we provided an Excel spreadsheet that is
freely available from the externally hosted online
supplementary material.[32]
To verify the predictions available through
equation (3), we performed the NPase-catalyzed
phosphorolysis of uridine (1) and adenosine (5) at
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
and chelating agents on the equilibrium states of
nucleoside phosphorolysis may be explored
experimentally by future studies.
Experimental Section
General Remarks
All chemicals were of analytical grade or higher and
purchased, if not stated otherwise, from Sigma Aldrich
(Steinheim, Germany), Carbosynth (Berkshire, UK), Carl
Roth (Karlsruhe, Germany), TCI Deutschland (Eschborn,
Germany) or VWR (Darmstadt, Germany). All nucleosides
(Figure S1) and nucleobases were used without prior
purification. Water deionized to 18.2 MΩ∙cm with a Werner
water purification system was used. For the preparation of
NaOH solutions deionized water was used.
Figure 5. Predicted (pred.) and experimental (exp.)
phosphorolytic conversion of uridine (1) and adenosine (5).
Reactions were performed with 2 mM substrate and 5, 10,
20 or 40 mM (2.5, 5, 10 or 20 eq.) K2HPO4 and 40 µg∙ml-1
Py-NPase Y02 or 40 µg∙ml-1 Pu-NPase N02 in 50 mM
glycine buffer at pH 9 and 40 °C in a total volume of 500 µL.
Samples were taken after 10, 15 and 20 min to confirm
equilibrium. The datapoints show the average of the three
equilibrium data points. The predictions were calculated
with equation (3) using 퐾′(T) obtained through the ∆푅퐻′∗
and ∆푅푆′∗ data for these substrates.
Enzymes
Enzymes were either purchased from BioNukleo GmbH
(Berlin, Germany) or Sigma Aldrich (see Table S2).
Enzymes provided by BioNukleo were E-PyNP-0001 (Py-
NPase Y01; EC 2.4.2.2), E-PyNP-0002 (Py-NPase Y02; EC
2.4.2.2), E-PNP-0001 (Pu-NPase N01; EC 2.4.2.1), E-PNP-
0002 (Pu-NPase N02; EC 2.4.2.1), E-UP-0001 (Escherichia
coli uridine phosphorylase; E. coli UP; EC 2.4.2.3), E-TP-
0001 (E. coli thymidine phosphorylase; E. coli TP; EC
2.4.2.4) and E-PNP-04 (E. coli purine nucleoside
phosphorylase; E. coli Pu-NPase; EC 2.4.2.1). Enzymes
were desalted against 2 mM KH2PO4 (pH 7.0, measured at
25 °C) buffer and stored at 4 °C at concentrations listed in
Table S2. Bacillus subtilis pyrimidine phosphorylase (B.
subtilis Py-NPase; EC 2.4.2.2) was obtained from Sigma
Aldrich and prepared as a 1 mg∙ml-1 solution in 2 mM
KH2PO4 buffer (pH 7.5, measured at 25 °C) prior to use.
Conclusion
In the present work, we broadly demonstrated that
nucleoside phosphorolysis catalyzed by NPases is a
thermodynamically controlled endothermic reversible
equilibrium reaction. Therefore, maximum yields for
each substrate are independent of the NPase used, as
demonstrated by several examples, and can be
predicted via the substrate-specific apparent
equilibrium constant 퐾′. We anticipate that this holds
true for all nucleosides that can be subjected to
phosphorolysis. Furthermore, we presented data on the
temperature-dependency of the equilibrium constants
of phosphorolysis of 24 nucleosides that display
characteristic behavior. The available data allow for
the calculation of 퐾′ at any given temperature and
enable accurate prediction of phosphorolysis or
transglycosylation yields for a range of pyrimidine and
purine nucleosides.[33] Subsequently, the equations
described by Alexeev et al.[19], the thermodynamic
data reported in this study, as well as the tools provided
herein (see external supplementary material)[32] serve
to facilitate streamlined reaction engineering of
NPase-catalyzed reactions by in silico yield prediction
and thermodynamic reaction control.[33]
Since our findings show that maximum yields in
NPase-catalyzed reactions can be achieved
independently of the enzyme applied, we believe that
efforts to influence the yield of these reactions by
varying the enzyme are unlikely to bear fruit. Instead,
extending the toolbox of available NPases to improve
kinetic parameters to reach reaction equilibrium faster
or to convert challenging substrates such as arabinosyl
nucleosides, fluorinated glycosides or bulky
nucleobases will certainly prove beneficial.
The activity of the stock solutions of Py-NPase Y02 and Pu-
NPase N02 was assessed by the UV/Vis spectroscopy-based
method described recently (also see below).[34] Reactions
were performed with 1 mM of nucleoside substrate in
50 mM glycine buffer and 50 mM K2HPO4 at pH 9 and
40 °C. One unit (U) is defined as the conversion of 1 µmol
of substrate per minute. For Py-NPase Y02 and Pu-NPase
N02 an activity of 40 U∙mg-1 (65 U∙ml-1) and 57 U∙mg-1
(379 U∙ml-1) for their natural substrates uridine (1) and
adenosine (5), respectively, was determined.
Phosphorolysis of pyrimidine and purine nucleosides
Enzymatic nucleoside phosphorolysis reactions were
prepared from stock solutions of nucleoside, phosphate,
buffer and water in 1.5 mL reaction tubes (Sarstedt,
Nümbrecht, Germany) and started by the addition of the
enzyme. Reactions were performed in duplicate with 2 mM
nucleoside and 10 mM K2HPO4 in 50 mM MOPS buffer
(pH 7.5, measured at 30 °C) in a total volume of 500 µL.
Prior to the addition of enzyme solution, reactions were
preheated to 37 °C. Unless stated otherwise, final enzyme
concentrations of 10 µg∙ml-1 were used for all enzymes and
substrates. For the phosphorolysis of 5-fluorouridine (3)
with Pu-NPase N02, 600 µg∙ml-1 of enzyme were employed.
To investigate possible effects of the enzyme concentration,
uridine (1) was subjected to phosphorolysis using 10—
40 µg∙ml-1 of Py-NPase Y02. To exclude possible enzyme
inactivation effects, 1 was also subjected to phosphorolysis
with 40 µg∙ml-1 of Py-NPase Y02 (20 µg total enzyme) and
10 µg of the enzyme were added after apparent reaction
completion at 10 min and 15 min each. To validate the
predictions of phosphorolysis conversion with different
phosphate concentrations, 2 mM of 1 and 5, respectively,
were converted with 40 µg∙ml-1 Py-NPase Y02 or 40 µg∙ml-1
Pu-NPase N02 in 50 mM glycine buffer at pH 9 and 40 °C
with 5, 10, 20 or 40 mM (2.5, 5, 10 or 20 eq.) K2HPO4. The
Lastly, the quantification of effects of reaction
conditions such as ionic strength, different salts, pH
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
reactions were monitored, and equilibrium samples were
taken after 10, 15 and 20 min.
Acknowledgements
Monitoring of enzyme reactions
We would like to thank Sarah von Westarp for her support with
enzyme preparation, Niels Krausch for initial support with
software development, and Dr. Erik Wade for revision of the
manuscript. We are grateful for the support of R.T.G. by the
German Research Foundation (DFG) under Germany´s
Excellence Strategy – EXC 2008/1– 390540038 (UniSysCat), and
by the Einstein Foundation Berlin (ESB) - Einstein Center of
Catalysis (EC2).
Sampling, data collection and analysis were carried out as
described previously.[34] Briefly, samples of 30 µL (for
pyrimidine nucleosides) or 20 µL (for purine nucleosides)
were withdrawn from the reaction mixture and pipetted into
100 mM NaOH solution (200 mM NaOH solution for 3, 4
and 13—16) to give a final volume of 500 µL of diluted
alkaline sample. From this mixture, 200 µL were pipetted
into wells of a UV/Vis-transparent 96-well plate (UV-STAR
F-Bottom #655801, Greiner Bio-One). The UV/Vis
absorption spectra of these alkaline samples were recorded
from 250 to 350 nm to determine the nucleoside/nucleobase
ratio via spectra unmixing. All data presented in this study
can be obtained from an external online repository[32] along
with the software for spectral unmixing and metadata
treatment detailed in our previous work.[34,35]
References
[1] L. P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet,
Nat. Rev. Drug Discov. 2013, 12, 447–464.
[2] J. Shelton, X. Lu, J. A. Hollenbaugh, J. H. Cho, F.
Amblard, R. F. Schinazi, Chem. Rev. 2016, 116, 14379–
14455.
[3] S. Kamel, H. Yehia, P. Neubauer, A. Wagner, in Enzym.
Chem. Synth. Nucleic Acid Deriv., 2019, pp. 1–28.
[4]H. Yehia, S. Kamel, K. Paulick, A. Wagner, P. Neubauer,
Curr. Pharm. Des. 2017, 23, 6913–6935.
[5] S. Kamel, M. Weiß, H. F. T. Klare, I. A. Mikhailopulo,
P. Neubauer, A. Wagner, Mol. Catal. 2018, 458, 52–59.
[6] I. V. Kulikova, M. S. Drenichev, P. N. Solyev, C. S.
Alexeev, S. N. Mikhailov, Eur. J. Org. Chem. 2019,
6999–7004.
[7] I. V. Fateev, K. V. Antonov, I. D. Konstantinova, T. I.
Muravyova, F. Seela, R. S. Esipov, A. I. Miroshnikov, I.
A. Mikhailopulo, Beilstein J. Org. Chem. 2014, 10,
1657–1669.
[8] H. Komatsu, T. Araki, Nucleosides. Nucleotides Nucleic
Acids 2005, 24, 1127–1130.
[9] I. D. Konstantinova, K. V. Antonov, I. V. Fateev, A. I.
Miroshnikov, V. A. Stepchenko, A. V. Baranovsky, I. A.
Mikhailopulo, Synthesis. 2011, 1555–1560.
[10] M. Rabuffetti, T. Bavaro, R. Semproli, G. Cattaneo, M.
Massone, F. C. Morelli, G. Speranza, D. Ubiali,
Catalysts 2019, 9, 355.
[11] B. Z. Eletskaya, D. A. Gruzdev, V. P. Krasnov, G. L.
Levit, M. A. Kostromina, A. S. Paramonov, A. L.
Kayushin, I. S. Muzyka, T. I. Muravyova, R. S. Esipov,
et al., Chem. Biol. Drug Des. 2019, 93, 605–616.
[12] X. Zhou, K. Szeker, B. Janocha, T. Böhme, D.
Albrecht, I. A. Mikhailopulo, P. Neubauer, FEBS J.
2013, 280, 1475–1490.
Temperature dependence of the equilibrium constant
Equilibrium constants for the phosphorolysis of 24
nucleosides (1—24) were determined at pH 9 and at 40, 50,
60 and 70 °C. All reactions were performed with 2 mM
nucleoside substrate and 10 mM K2HPO4 in 50 mM glycine
buffer at pH 9. Unless stated otherwise, 16 µg∙mL-1
(0.6 U∙mL-1) Py-NPase Y02 (for pyrimidine nucleosides) or
66 µg∙mL-1 (3.7 U∙mL-1) Pu-NPase N02 (for purine
nucleosides) were applied. For inosine (23) and 2’-
deoxyinosine (24) 330 µg∙mL-1 of Pu-NPase N02 were used
and for 5-iodouridine (13) and 2’-deoxy-5-iodouridine (14)
32 µg∙mL-1 of Py-NPase Y02. All reactions were performed
in duplicate at temperatures from 40—70 °C in steps of
10 °C. Three samples were taken from each reaction once
equilibrium was reached to confirm reaction completion.
The time points used for sampling are given in Table S3. In
total, 576 data points were recorded. Outliers that either
displayed an elevated baseline due to UV absorption of the
96-well plate or differed more than 1.5 percentage points
from the other two data points within a sample set of a given
temperature and reaction mixture were excluded from data
interpretation. Consequently, 519 data points were
considered for further evaluation. Data treatment and fitting
was carried out with Origin ProLab 9. All data, as well as
all subsequent calculations can be inspected as freely
available data from an external online repository.[32] For
each data point, 퐾′ was calculated via equation (1) and
transformed apparent reaction enthalpy ∆푅퐻′∗ [J∙mol-1] and
transformed apparent reaction entropy ∆푅푆′∗ [J∙mol-1∙K-1]
were fitted directly to all available datapoints for a substrate
with equation (2). Similarly, ∆푅퐺′ was calculated with
equation (2) assuming that the apparent values ∆푅퐻′∗ and
∆푅푆′∗ are independent of the reaction temperature in the
considered range. The errors ∆∆푅퐻′∗ [J∙mol-1], and ∆∆푅푆′∗
[J∙mol-1∙K-1] were derived from the fit and ∆∆푅퐺′ was
calculated via Gaussian error propagation:
[13] K. Szeker, X. Zhou, T. Schwab, A. Casanueva, D.
Cowan, I. A. Mikhailopulo, P. Neubauer, J. Mol. Catal.
B Enzym. 2012, 84, 27–34.
2
2
′
′∗
∆∆푅퐺 = ∆∆푅퐻 + 푇2 ∆∆푅푆′∗
(4)
√
[14] I. Serra, T. Bavaro, D. A. Cecchini, S. Daly, A. M.
Albertini, M. Terreni, D. Ubiali, J. Mol. Catal. B Enzym.
2013, 95, 16–22.
with definitions from above.
[15] D. F. Visser, F. Hennessy, J. Rashamuse, B. Pletschke,
D. Brady, J. Mol. Catal. B Enzym. 2011, 68, 279–285.
[16] D. P. Nannemann, K. W. Kaufmann, J. Meiler, B. O.
Bachmann, Protein Eng. Des. Sel. 2010, 23, 607–616.
[17] X. Xie, W. Huo, J. Xia, Q. Xu, N. Chen, Enzyme
Microb. Technol. 2012, 51, 59–65.
Conflict of Interest
A.W. is CEO of the biotech company BioNukleo GmbH.
F.K. is a researcher at BioNukleo GmbH and P.N. is a
member of the advisory board.
[18] R. T. Giessmann, N. Krausch, F. Kaspar, N. M. Cruz
Bournazou, A. Wagner, P. Neubauer, M. Gimpel,
Processes 2019, 7, 380.
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
[19] C. S. Alexeev, I. V. Kulikova, S. Gavryushov, V. I.
Tararov, S. N. Mikhailov, Adv. Synth. Catal. 2018, 360,
3090–3096.
[20] IUPAC, Compendium of Chemical Terminology,
Blackwell Scientific Publications, Oxford, 1997.
[21] N. G. Panova, E. V. Shcheveleva, C. S. Alexeev, V. G.
Mukhortov, A. N. Zuev, S. N. Mikhailov, R. S. Esipov,
D. V. Chuvikovsky, A. I. Miroshnikov, Mol. Biol. 2004,
38, 770–776.
[28] M. Camici, F. Sgarrella, P. L. Ipata, U. Mura, Arch.
Biochem. Biophys. 1980, 205, 191–197.
[29] A. Hatano, A. Harano, Y. Takigawa, Y. Naramoto, K.
Toda, Y. Nakagomi, H. Yamada, Bioorg. Med. Chem.
2008, 16, 3866–3870.
[30] J. C. Leer, K. Hammer-Jespersen, M. Schwartz, Eur. J.
Biochem. 1977, 75, 217–224.
[31] G. A. Kicska, P. C. Tyler, G. B. Evans, R. H. Furneaux,
K. Kim, V. L. Schramm, J. Biol. Chem. 2002, 277,
3219–3225.
[22] R. A. Alberty, Biophys. Chem. 2007, 127, 91–96.
[23] A. Vita, C. Y. Huang, G. Magni, Arch. Biochem.
Biophys. 1983, 226, 687–692.
[32] F.
Kaspar,
R.
T.
Giessmann,
2019,
10.5281/zenodo.3530526
[24] R. Bose, E. W. Yamada, Biochemistry 1974, 13, 2051–
2056.
[25] R. A. Alberty, J. Chem. Thermodyn. 2004, 36, 593–
601.
[26] R. N. Goldberg, Y. B. Tewari, T. N. Bhat, J. Phys.
Chem. Ref. Data 2007, 4, 1347–1397.
[33] F. Kaspar, R. T. Giessmann, K. Hellendahl, P.
Neubauer, A. Wagner, M. Gimpel, manuscript in
preparation.
[34] F. Kaspar, R. T. Giessmann, N. Krausch, P. Neubauer,
A. Wagner, M. Gimpel, Methods Protoc. 2019, 2, 60.
[35] R.
T.
Giessmann,
N.
Krausch,
2019,
[27] T. A. Krenitsky, G. W. Koszalka, J. V. Tuttle,
Biochemistry 1981, 20, 3615–3621.
10.5281/zenodo.3243376.
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901230
Advanced Synthesis & Catalysis
FULL PAPER
Thermodynamic Reaction Control of Nucleoside
Phosphorolysis
Adv. Synth. Catal. Year, Volume, Page – Page
Felix Kaspar, Robert T. Giessmann, Peter
Neubauer, Anke Wagner* and Matthias Gimpel
10
This article is protected by copyright. All rights reserved.