Efficient biocatalytic synthesis of dihalogenated purine nucleoside analogues applying thermodynamic calculations

Article abstract of DOI:10.3390/molecules25040934

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

Full text of DOI:10.3390/molecules25040934

molecules
Article
Efficient Biocatalytic Synthesis of Dihalogenated
Purine Nucleoside Analogues Applying
Thermodynamic Calculations
Heba Yehia 1,2,† , Sarah Westarp 1,3,† , Viola Röhrs 4, Felix Kaspar 1,3
,
Robert T. Giessmann 1 , Hendrik F.T. Klare 5 , Katharina Paulick 1, Peter Neubauer 1
,
Jens Kurreck 4 and Anke Wagner 1,3,
*
1
Chair of Bioprocess Engineering, Faculty III Process Sciences, Institute of Biotechnology, Technische
Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany; HEBAYEHYA@hotmail.com (H.Y.);
sarah.westarp@bionukleo.com (S.W.); f.kaspar@tu-braunschweig.de (F.K.); rgiessmann@gmail.com (R.T.G.);
katharina.paulick@tu-berlin.de (K.P.); peter.neubauer@tu-berlin.de (P.N.)
Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre, Dokki, 12622 Cairo, Egypt
2
3
4
BioNukleo GmbH, Ackerstr. 76, 13355 Berlin, Germany
Chair of Applied Biochemistry, Faculty III Process Sciences, Institute of Biotechnology, Technische
Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany; viola.roehrs@tu-berlin.de (V.R.);
jens.kurreck@tu-berlin.de (J.K.)
Faculty II Mathematics and Natural Sciences, Institute of Chemistry, Technische Universität Berlin,
Strasse des 17. Juni 135, 10623 Berlin, Germany; hendrik.klare@tu-berlin.de
Correspondence: anke.wagner@tu-berlin.de; Tel.: +49-30-314-72183
5
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 January 2020; Accepted: 18 February 2020; Published: 19 February 2020
Abstract: The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and
efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues
were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using
uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields
were calculated after the determination of equilibrium constants through monitoring the equilibrium
conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were
comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product
yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed.
Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product
were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in
positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of
dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the
inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than
with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken
together, we demonstrate that thermodynamic calculations and small-scale experiments can be used
to produce nucleoside analogues with high yields and purity on larger scales. The procedure can
be used for the generation of new libraries of nucleoside analogues for screening experiments or to
replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.
Keywords: cytostatics; dihalogenated nucleoside analogue; yield prediction; thermostable nucleoside
phosphorylase; thermodynamic calculations; leukemic cell line
Molecules 2020, 25, 934; doi:10.3390/molecules25040934
www.mdpi.com/journal/molecules

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1. Introduction
The standard production route for nucleoside analogues in the pharmaceutical industry is still
chemical synthesis. It usually involves protection–deprotection steps and the use of hazardous
chemicals and solvents. Furthermore, chemical synthesis routes for purine nucleoside analogues
usually show low selectivity and finally lead to low product yields [
of chemical synthesis routes, alternative methods have been developed. Enzymatic synthesis is
environmentally more friendly, highly selective, and efficient [ ]. The most commonly applied
enzyme-catalyzed reaction for the preparation of nucleosides and their analogues is the one-pot
transglycosylation with pyrimidine and purine nucleoside phosphorylases [ ]. Here, the sugar moiety
1,2]. Due to the drawbacks
3
4
is exchanged between two nucleobases in the presence of inorganic phosphate.
So far, mainly nucleoside phosphorylases (NP) of mesophilic organisms have been used for the
synthesis of nucleoside analogues. However, enzymes from thermophilic bacteria or archaea are of
increasing interest, as they are active over a wide temperature and pH range, which facilitates the
solubility of substrates. Furthermore, mass transfer is improved due to a decreased viscosity and an
increase in diffusion coefficient and flux at higher temperatures [5,6]. In addition, due to the ease of
their purification, thermophilic enzymes are cost-effective biocatalysts for industrial processes [6].
Nucleoside analogues form a valuable class of drugs that have been widely used for more than
50 years for several indications such as cancer, viral, or protozoal infections [
analogues such as cladribine, fludarabine, or clofarabine are marketed drugs for the treatment of
hematological malignancies [ ]. These compounds are derivatives of adenosine and show modifications
7–9]. Purine nucleoside
8
in the base and/or the sugar moiety. Cladribine, fludarabine, and clofarabine have a halogen atom at
position 2 of the nucleobase, which was shown to be an important prerequisite for deaminase resistance
and, therefore, its increased intracellular activity [10]. These compounds all share a similar mechanism
of action, such as the inhibition of DNA synthesis, inhibition of DNA repair, and accumulation of DNA
strand breaks.
Biological properties of enzymatically produced nucleoside analogues have been studied before.
Analogues of 20-deoxyribosides were produced using mesophilic nucleoside deoxyribosyltransferase
from Lactobacillus leichmannii [11,
12]. In a first attempt, 12 analogues of 20-deoxyadenosine were
enzymatically produced at a 100 to 400 mg scale with an average yield of 64% [11]. In a second study,
8-substituted purine nucleoside analogues were synthesized with yields of <10 to 70% [12]. Of the
compounds produced, 2-chloro and 2-bromo analogues of 20-deoxyadenosine were good inhibitors in
tumor cell lines [11,12].
In the present study, the enzymatic synthesis of 2,6-dichloropurine riboside (3a), 2,6-dichloropurine
deoxyriboside (3b), 6-chloro-2-fluoropurine riboside (3c), and 6-chloro-2-fluoropurine deoxyriboside
(3d) with thermostable nucleoside phosphorylases was optimized based on thermodynamic equilibrium
state calculations [13]. Equilibrium constants for dihalogenated nucleosides were calculated and used
to determine optimum reactions to reach 90% or 95% product yields. The transferability of the results
obtained on an analytical scale to up-scaling experiments was analyzed. Furthermore, we evaluated
the cytotoxic activity of dihalogenated nucleoside analogues in a hematologic tumor cell line. While
2-halogenated nucleoside analogues were shown to have an increased stability and activity due to a
resistance to deaminases [10], dihalogenated compounds have not been studied before.
2. Results
2.1. Optimization of the Synthesis of Dihalogenated Nucleosides Based on Thermodynamic Calculations
Transglycosylation reactions were employed to produce 2,6-dichloropurine (2a) and
6-chloro-2-fluoropurine (2b) containing nucleosides using uridine (1a) and thymidine (1b) as sugar
donors (Scheme 1). Pyrimidine nucleoside phosphorylase (PyNP, EC 2.4.2.2) and purine nucleoside
phosphorylase (PNP, EC 2.4.2.1) from a thermophilic organism were applied as biocatalysts.

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R3
N
O
R1
O
N
N
NH
R3
N
N
R1
NH
R4
HO
O
HO
O
N
N
O
N
PyNP, PNP, Pi
O
N
H
+
+
R4
N
H
R2
HO
R2
HO
3a: R2=OH, R3=Cl, R4=Cl
3b: R2=H, R3=Cl, R4=Cl
3c: R2=OH, R3=Cl, R4=F
3d: R2=H, R3=Cl, R4=F
1a: R1=H, R2=OH
1b: R1=CH3, R2=H
2a: R3=Cl, R4=Cl
2b: R3=Cl, R4=F
4a: R1=H
4b: R1=CH3
Scheme 1.
using thermostable nucleoside phosphorylases as biocatalysts and uridine
thymidine 1b as sugar donors. 2a—2,6-dichloropurine, 2b—6-chloro-2-fluoropurine,
Transglycosylation reaction to produce dihalogenated purine nucleosides
(
1a) and
(
)
3a—2,6-dichloropurine riboside, 3b—2,6-dichloropurine deoxyriboside, 3c—6-chloro-2-fluoropurine
riboside, 3d—6-chloro-2-fluoropurine deoxyriboside, 4a—uracil, 4b—thymine, PyNP—pyrimidine
nucleoside phosphorylase, PNP—purine nucleoside phosphorylase, Pi—inorganic phosphate.
To efficiently produce 2a- and 2b-containing nucleosides in a transglycosylation reaction,
equilibrium state thermodynamic calculations were carried out [13]. We have recently shown
that nucleoside phosphorolysis is a reversible endothermic reaction under tight thermodynamic
control [14]. Consequently, it is possible to predict the equilibrium states of transglycosylation reactions,
if the equilibrium constants of phosphorolysis of both participating nucleosides are known [13].
However, for 3a
equilibrium states of analytical-scale reactions were used to calculate these values for 3a
previously published equations (for more detail, please see reference [14], the respective Supplementary
Materials [15 16], as well as the externally hosted supplementary material of this publication [17]).
The recently described equilibrium constants of the sugar donors 1a and 1b 14] served as the basis for
–
d, equilibrium constants of phosphorolysis have not been described before. Thus, the
–
d, employing
,
[
these calculations. Analytical-scale reactions were performed with equal concentrations of base and
sugar donor of 5 mM and 2 mM of phosphate. At equilibrium, product formation was between 55%
(3b) and 60% (3a and 3c).
Thus, the calculated equilibrium constants (K2) were in the range of 0.071 (3d) to 0.081 (3a) (Table 1).
These values fit well with previously reported equilibrium constants of the phosphorolysis of other
purine nucleosides [14]. Equilibrium constants were slightly higher for 2a-derived nucleosides (3a and
3b) compared to 2b-derived nucleosides (3c and 3d) and for deoxyribonucleosides in comparison to
ribonucleosides (Table 1).
Table 1. Equilibrium state thermodynamic calculations were used to determine appropriate reaction
conditions for the maximum conversion of 3a–d.
Product Formation
[%] at Equilibrium *
Sugar Donor Excess to Reach:
90% Product Yield 95% Product Yield
Equilibrium
Constant K2 **
Product
3a
3b
3c
60
55
60
57
0.076
0.081
0.076
0.071
4.5 9.0
6.1
4.4
5.4
12.5
8.8
11.0
3d
* Experiments were performed with 5 mM nucleobase, 5 mM sugar donor 1a or 1b, and 2 mM potassium phosphate
buffer at 40 ◦C to determine the equilibrium constants. ** Equilibrium constants of phosphorolysis of the target
compounds were calculated as described in [13]. Known equilibrium constants for these sugar donors at 40 ◦C were
employed for the calculations [14].
Employing these equilibrium constants, optimal reaction conditions to provide 90% or 95%
conversion were calculated. Sugar donor excess was predicted to be in the range of 4.4 to 6.1 to reach
90% product yield and 8.8 to 12.5 to obtain 95% product formation (Table 1). In accordance with the

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equilibrium constants of 1a and 1b, a higher sugar donor excess was estimated to be needed for the
synthesis of deoxyribonucleosides compared to the corresponding ribonucleosides.
Analytical-scale experiments with the selected conditions confirmed the theoretical predictions.
Product formation of 90% and 95% was observed under the tested reaction conditions for all target
compounds (Figure 1). However, the time to reach equilibrium differed between ribonucleosides and
deoxyribonucleosides. While equilibrium was reached within 1 h for 3b and 3d, equilibrium was
only observed after 6 h for 3a and 3c (Figure 1). Therefore, for semi-preparative-scale, reactions
with ribonucleosides and deoxyribonucleosides were stopped after 6 h and 2 h, respectively.
In semi-preparative experiments, conditions were used to reach 90% product yield. Doubling
of the amount of 1a and 1b would have been needed to reach 95% product formation. However, high
quantities of the sugar donor would increase the substrate cost and the ecological burden, as a lot of
sugar donor is wasted. In addition, it complicates purification, as an efficient product separation is no
longer ensured.
Figure 1. Synthesis of 3a (A), 3b (B), 3c (C), and 3d (D) in transglycosylation reactions at small
and semi-preparative scale using thermostable nucleoside phosphosporylases PyNP 02 and PNP 02.
Equilibrium state calculations served as a basis to determine reaction conditions and were confirmed
by reactions performed at small and semi-preparative scale. Base concentrations of 5 mM and sugar
donor concentrations of 22 to 62.5 mM (Table S3) were used. Substrate concentrations were chosen
based on thermodynamic calculations. Thermostable nucleoside phosphorylases were added to a
final concentration of 0.1 mg mL−1 each. Enzymatic reactions were performed in 0.5 mM potassium
phosphate buffer at pH 7.5 using a reaction temperature of 40 ◦C. Conversion was calculated based on
formation of the product nucleoside from the halogenated base.
2.2. Synthesis of Dihalogenated Nucleosides in Semi-Preparative Scale
Dihalogenated nucleosides were produced at 50-mL scale to validate the efficiency and specificity
of the synthesis and purification process. Products were purified by HPLC. Recovery of the sugar
donor was validated for 3b as an example. Appropriate reaction conditions were employed to reach
90% product yield.

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Enzymatic transglycosylation reactions led to conversions of 83% (3a), 92% (3b), 90% (3c), and
92% (3d) (Figure 1, Table S1). This was in good accordance with small-scale experiments. The time
course of product formation was also well comparable with small-scale studies. Equilibrium was
reached within 1 h for 3b and 3d and mainly within 6 h for 3a and 3c (Figure 1). Except for the expected
reaction products uracil (4a) or thymine (4b), no further by-products were observed by HPLC analysis
in any of the reactions.
Products synthesized in transglycosylation reactions were purified by semi-preparative HPLC.
Proteins were removed by filtration as a prerequisite for this step. Product loss during protein removal
and HPLC purification was in the range of 18% (3a) to 31% (3b and 3d) (Table S1). Still, this step was
required to guarantee high product purity. Freeze-drying yielded the target compounds as white
powders. The highest losses in product quantity (28% to 32%) were observed in this step (Table S1).
Losses can be reduced by collecting more of the product during HPLC purification and by conducting
a second round of freeze-drying.
In total, 34 mg, 36 mg, 32 mg, and 32 mg of purified powder were obtained for 3a, 3b, 3c, and 3d,
respectively (Table S1). These values correspond to a recovery of approximately 50% for each target
compound (Table S1). Product purity was greater than 96%, and structures were confirmed by NMR
(Table S2, Figure S1 and Figure S2).
Since the sugar donors used can be a cost factor for the enzymatic synthesis of nucleoside
analogues, the recovery of 1b was analyzed using the synthesis of 3b as an example. In the enzymatic
reaction, 369 mg of 1b was added. The transglycosylation reaction resulted in 74 mg (20%) conversion
to 4b. After protein removal and HPLC purification, 251 mg of the remaining 1b were recovered. In the
freeze-drying process 22% of 1b was lost and in total, 195 mg 1b (53% yield) was recaptured from the
initial 369 mg added as the starting material.
2.3. Effect of Dihalogenated Nucleoside Analogues on Cell Growth
Dihalogenated nucleosides 3a–d were tested for their ability to inhibit the growth of HL-60 cells
in culture. Monohalogenated cladribine, which is known to efficiently inhibit HL-60 cells [18
used as positive control. While for cladribine an IC50 value of 0.22 M [ 0.11 M] was determined,
50% of inhibition by the dihalogenated nucleoside analogues was observed for concentrations between
10 and 100 M (Figure 2). The inhibition of growth of the control cell line HEK293 was in the similar
,19], was
µ
±
µ
µ
range of nucleoside analogue concentrations. No unspecific growth inhibition in the control cell line
HEK293 was detected for cladribine. Hence, nucleoside analogues with halogen atoms in positions 2
and 6 are no suitable inhibitors of leukemia cell lines.
Figure 2. Biological activity of dihalogenated nucleoside analogues in the leukemia cell line HL-60 (
or the control cell line HEK293 ( ). Nucleoside analogues were serially diluted, and the percentage of
killed cells was determined by XTT assay.
A)
B

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3. Discussion
The current study shows the feasibility of optimizing the enzymatic synthesis of modified
nucleosides based on initial experiments and thermodynamic calculations to determine equilibrium
constants. Dihalogenated nucleoside analogues 3a–d were formed with percentages of conversion of
90% to 95% at small scale and 83% to 92% at semi-preparative scale. Yields of purified product were
approximately 50%.
Dihalogenated purine bases have been studied before as substrates for NPs from extreme
environments. Both 2a and 2b were converted to the respective riboside and deoxyriboside nucleosides
by a thermostable PNP from Geobacillus thermoglucosidasius with conversions of 54% to 69% [20].
A sugar donor-to-nucleobase ratio of 3.34 in the presence of 3.34 equivalents of phosphate (compared
to the base concentration) was used. Using an immobilized PyNP of Thermus thermophilus and the
same PNP, Zhou and colleagues later observed increasing product formation with reduced phosphate
concentrations [21]. A negative impact on the product yield of phosphate equivalents above 0.3 in
comparison to the nucleobase was recently confirmed by thermodynamic studies on transglycosylation
reactions [13,22]. The advantages of the application of thermodynamic equilibrium state calculations
in transglycosylation reactions is convincingly confirmed in the present study, which also represents
the first example of the fruitful application of this methodology in practice. After calculating the
equilibrium constants based on a single analytical reaction each, reaction conditions were designed to
reach product yields of 90% and 95%. Small- and semi-preparative-scale experiments employing the
improved conditions confirmed the calculated predictions. Hence, under optimized conditions, higher
product yields were observed compared to previous studies [20,21].
Besides the optimization of the enzymatic synthesis process by thermodynamic calculation,
the present study also investigated the impact of a chlorine atom at position 6 of the nucleoside
with respect to its inhibitory effect on leukemic cells. This was based on the observation that
halogenation is an important prerequisite for anticancer activity [
the compound susceptible to deamination by adenosine deaminase (ADA) [25
chlorine or fluorine atom in position 2 of adenosine strongly increased its stability, as observed for
the very potent leukemia drugs cladribine and fludarabine [23 26]. Interestingly, the dihalogenated
3,23
,24], since amino groups make
,26]. The addition of a
,
analogues tested in the present study with cell line HL-60 showed less growth inhibition than the
monohalogenated cladribine. An explanation for the low activity could involve the uptake of the
dihalogenated nucleosides, the activation by nucleoside and nucleotide kinases, or the interaction with
the intracellular targets. The uptake of cladribine involves equilibrative nucleoside transporter 1 and
2 as well as concentrative nucleoside transporter 3 [27], which might not recognize dihalogenated
nucleosides. Most of the known nucleoside analogue drugs including cladribine are only active as the
respective di- or triphosphorylated nucleotides, and activation in vivo is often insufficient [28]. Hence,
a lack of activity might be explained by human kinases not efficiently phosphorylating dihalogenated
nucleosides. Intracellular targets of cladribine have been recently well summarized by Freyer and
colleagues [29]. Ribonucleotide reductase is one of the main targets of cladribine. Its inhibition leads
to a depletion of deoxyadenosine and hence to an imbalance within the deoxyribonucleotide pool.
This leads to a number of other effects such as endonuclease activation and double-strand DNA
breaks. Additionally, the inhibition of adenosine deaminase [30] or RNA polymerase [31] by cladribine
were described before. However, further studies are needed to better understand the limitations of
dihalogenated nucleosides in the inhibition of leukemia cell lines.
Although the dihalogenated nucleoside analogues showed less growth inhibition in leukemia cell
line HL-60 than their monohalogenated counterparts, they may also serve as valuable starting material
for the preparation of a broad palette of modified nucleosides [32,33]. Dihalogenated nucleoside
analogues have a much higher solubility than monohalogenated analogues [20] and can therefore
serve as precursors for the synthesis of new compounds or approved substances in industrial scale.

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4. Materials and Methods
4.1. General Information
All chemicals and solvents were of analytical grade or higher and purchased, if not stated otherwise,
from Sigma-Aldrich (Steinheim, Germany), Carl Roth (Karlsruhe, Germany), TCI Deutschland
(Eschborn, Germany), and VWR (Darmstadt, Germany). Cladribine was obtained from TCI with
a purity >98%. Water was purified and deionized by an EASYpure II purification system to reach
◦
a resistivity of 18.2 M
Ω
cm at 25 C (Werner, Leverkusen, Germany). Thermostable nucleoside
phosphorylases (PyNP 02 [E-PyNP-0002] and PNP 02 [E-PNP-0002]) were provided by BioNukleo and
used as recommended by the manufacturer. Nucleoside phosphorylases were heterologously expressed
in E. coli and originate from thermophilic bacteria with temperature optima at 60 ◦C. Enzymes were
purified by affinity chromatography. Enzyme concentrations of 0.1 mg mL−1 were used for PyNP and
PNP. PyNP 02 and PNP 02 had activities of 40 U mL−1 and 60 U mL−1 at 40 ◦C and pH 9 for their natural
substrates uridine and adenosine, respectively, as determined by the UV spectroscopy-based assay
described recently [34]. Cell lines HEK-239 and HL-60 were purchased from the German Collection of
Microorganisms and Cell Cultures GmbH. All experimental and calculated data are available free of
charge from an external online repository [17].
4.2. Optimization of the Enzymatic Synthesis of Dihalogenated Purine Nucleosides
Thermodynamic calculations were shown to allow for a reliable calculation of product yields in
nucleoside phosphorylase catalyzed transglycosylation reactions based on equilibrium constants of
phosphorolysis [13
(
,22]. Equilibrium constants for the phosphorolysis of uridine (1a) and thymidine
1b) were previously described to be 0.18 and 0.125, respectively, at 40 ◦C [14]. To estimate equilibrium
constants K2 for the synthesis of the dihalogenated nucleoside analogues, enzymatic reactions were
performed with 5 mM of sugar donor (1a or 1b), 5 mM of the purine base 2a or 2b) in 2 mM potassium
phosphate (pH 7.5) using 0.1 mg mL−1 of the biocatalysts PyNP 02 and PNP 02. Reactions were
performed at 40 ◦C for 20 h. To analyze sugar donor cleavage and product formation, samples were
analyzed by HPLC. First, 30 µL were removed from the reaction mixture, and an equal volume of
methanol was added to stop the reaction. After centrifugation at 21,500 g for 10 min, the supernatant
was diluted 1:10 and transferred to HPLC vials.
Solving the system of equilibrium constraints given by the laws of mass action of the first and
second (reverse) phosphorolysis of the transglycosylation sequence for these values, as described
recently [13,14], yielded equilibrium constants of phosphorolysis for 3a–d. Based on these equilibrium
constants, the sugar donor excess needed to reach 90% and 95% product formation was calculated [13].
Sugar donor excess was calculated to be between 4.4 and 6.1 to reach 90% product yield and 8.8 and
12.5 to guarantee 95% product formation (Table S3). Reactions were performed to verify theoretical
calculations at 1 mL scale. Base concentrations of 5 mM and enzyme concentration of 0.1 mg mL−1
for PyNP 02 and PNP 02 were used in 0.5 mM potassium phosphate buffer (pH 7.5). The reaction
temperature was 40 ◦C. Regular samples were taken to analyze sugar donor cleavage and product
formation by HPLC. A 30 µL sample was removed from the reaction mixture, and an equal volume of
methanol was added to stop the reaction. After centrifugation at 21,500 g for 10 min, the supernatant
was diluted 1:10 and transferred to HPLC vials.
4.3. Enzymatic Production of Dihalogenated Purine Nucleosides
To produce dihalogenated nucleoside analogues, base concentrations of 5 mM and sugar donor
concentrations of 22 to 30.5 mM were applied in a reaction volume of 50 mL. Thermostable pyrimidine
and purine nucleoside phosphorylase PyNP 02 and PNP 02 were added in a concentration of 0.1 mg
mL−1 each. Enzymatic reactions were performed in 0.5 mM potassium phosphate buffer at pH 7.5
using a reaction temperature of 40 ◦C. Reactions were stirred at 100 rpm. Regular samples were taken
to monitor enzymatic reactions by HPLC as described above.

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The reactions were stopped by enzyme removal after 2 h and 6 h for deoxyriboside and riboside
nucleosides, respectively. Protein was removed by filtration using centrifugal filter units with a cut-off
of 30 kDa (Millipore, Darmstadt, Germany) at room temperature. Filtrate was stored at 4 ◦C until
purification by semi-preparative HPLC.
Analytical HPLC was performed to determine product quantity after each step. Product loss per
step was determined based on the product quantity of step 1 (e.g., after enzymatic synthesis) and the
product quantity of the following step (e.g., after protein depletion and HPLC).
4.4. Cell Culture
HEK293 cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (Biowest)
containing 10% fetal bovine serum, 2 mM glutamine, non-essential amino acids, 4.5 mg mL−1
glucose, and the antibiotics penicillin and streptomycin. HL-60 cells were cultured in RPMI 1640 w/o
l-Glutamine supplemented with 10% fetal bovine serum, 2 mM glutamine, and the antibiotics penicillin
and streptomycin. Cells were grown at 37 ◦C in a humidified atmosphere with 5% CO2.
4.5. XTT Cell Proliferation Assay
Cytotoxicity was measured by the XTT assay at 450 nm as described by the manufacturer (Roche
Diagnostics, Basel, Switzerland). Phosphate-buffered saline was used as negative control. For HL-60
and HEK293 cells, 1.2
×
105 cells in growth media were transferred to each well of a 96-well flat-bottom
plate. Subsequently, modified nucleosides were added in varying concentrations. XTT assay was
performed after 24 h for HL-60 and HEK293 cells, as described previously [35]. At least three replicates
for each nucleoside concentration were performed. All experiments were conducted for a minimum of
three independent experiments.
4.6. Analytical HPLC
Nucleosides and bases were analyzed by HPLC (
λ = 260 nm) using a reversed-phase Kinetex
EVO C18 column (Phenomenex, Aschaffenburg, Germany) at 25 ◦C with a flow rate of 1 mL min−1
.
Substrates and products were identified by comparing their retention times with those of authentic
standards. Quantification was performed using a six-step calibration from a serial dilution in the range
from 0 to 1 mM over the peak area.
Gradual elution was performed using eluent A (20 mM ammonium acetate) and eluent B (100%
acetonitrile). The following gradient was used: 97% A and 3% B; 10-min linear gradient to 60% A and
40% B. The initial condition (97% A and 3% B) was restored afterwards and maintained for 4 min.
Typical retention times were as follows: 1a: 3.2 min, 4a: 2.4 min, 1b: 4.7 min, 4b: 4 min, 3c: 7.5
min, 3d: 8.2 min, 2b: 7 min, 3a: 8.2 min, 3b: 8.8 min, and 2a: 7.6 min. The percentage of conversion of
nucleosides was calculated as described previously (Equation (1)) [36].
Conc. of the product [mM]
Conversion % =
× 100
(1)
Conc. of the product [mM] + Conc. of the substrate [mM]
4.7. Purification of Nucleoside Analogues by Semi-Preparative HPLC
Nucleoside analogues were purified at room temperature using a KNAUER HPLC system
equipped with a Smartline Detector 2600 and an AZURA P 2.1 L pump. A flow rate of 21.24 mL min−1
and a Kinetex®
5
µm EVO C18 250
×
21.2 mm RP column were used. Deionized water and acetonitrile
were used as eluents, while the gradient was modified from the analytical method as summarized in
Table S4. Fractions containing either the sugar donor or the product were collected.
Collected samples were dried using a Christ Gamma 1–20 freeze-dryer (Osterode am Harz,
Germany). Residual water was removed by drying at 50 ◦C in a drying cabinet (DRY-Line 112 Prime,
VWR) for 8 h.

Molecules 2020, 25, 934
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4.8. NMR Analysis
The NMR spectra for 1H, 13C, and 19F were recorded in DMSO-d6 (purchased from Eurisotop) on a
Bruker Avance III 500 MHz instrument. Chemical shifts are reported in parts per million (ppm) and
are referenced to the residual solvent resonance as the internal standard (1H NMR:
δ
= 2.50 ppm for
= 39.52 ppm for DMSO-d6) [37]. 19F NMR spectra are referenced in compliance
with the unified scale for NMR chemical shifts as recommended by the IUPAC stating the chemical shift
DMSO-d5; 13C NMR:
δ
relative to CCl3F [38]. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet
,
mc = centrosymmetric multiplet, br = broad signal), coupling constants (Hz), and integration.
5. Conclusions
The present study demonstrates the value of small-scale experiments combined with
thermodynamic equilibrium state calculations to predict conditions for the efficient synthesis of
nucleosides at a larger scale. This approach was used to produce 3a–d in an enzymatic transglycosylation
reaction. Upscaling of the reaction was successfully performed based on the theoretical predictions.
The presented strategy can be transferred to the synthesis of a wide range of nucleoside analogues
using nucleoside phosphorylases as biocatalysts.
Supplementary Materials: Table S1. Yields and purity of dihalogenated nucleosides. Yields were determined
after each step in the synthesis and purification process. Purity was determined by analytical HPLC. Base
concentrations of 5 mM and sugar donor concentrations of 22 to 62.5 mM (Supplementary Table S3) were applied
in a reaction volume of 50 mL. Table S2. NMR characteristics of dihalogenated purine nucleosides produced in the
present study. Table S3. Conditions used for the synthesis of dihalogenated purine nucleosides with a product
yield of 90% or 95%. Reaction temperature was 40 ◦C. Table S4. HPLC methods applied for the purification of the
dihalogenated nucleoside analogues. A flow rate 21.24 mL min−1 was employed. Acetonitrile (ACN) and water
were applied as solvents. Figure S1. NMR spectra for 3a (A, C) and 3b (B, D). A, B: 1H NMR (500 MHz, DMSO-d6),
C, D: 13C{1H} NMR (126 MHz, DMSO-d6); Figure S2. NMR spectra for 3c (A, C, E) and 3d (B, D, F). A, B: 1H NMR
(500 MHz, DMSO-d6); C, D: 13C{1H} NMR (126 MHz, DMSO-d6); E,F: 19F{1H} NMR (471 MHz, DMSO-d6)
Author Contributions: Conceptualization, H.Y., K.P., J.K., and A.W.; methodology, H.Y., S.W., H.F.T.K., R.T.G.,
F.K., and V.R.; software, F.K. and R.T.G.; validation, F.K., H.Y., S.W., H.F.T.K., and V.R.; formal analysis, H.Y.,
S.W., V.R., and A.W.; investigation, H.Y., J.K., and A.W.; writing—original draft preparation, H.Y. and A.W;
writing—review and editing, H.Y., F.K., A.W, J.K., and P.N.; visualization, A.W.; supervision, J.K., P.N., and A.W.;
project administration, P.N., J.K., and A.W.; funding acquisition, R.T.G. All authors have read and agree to the
published version of the manuscript.
Funding: 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).
Acknowledgments: We would like to thank Bernd Krostitz for his support with cell line cultivation, and Erik
Wade for revision of the manuscript. We gratefully acknowledge support from the TU Berlin open access funding.
Conflicts of Interest: A.W. is the CEO, P.N. is a board member, and F.K. and S.W. are scientific researchers in the
biotech company BioNukleo GmbH. The authors have no other relevant affiliations or financial interest in or
financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No
conflict of interest is known to the other authors.
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Sample Availability: Samples of the compounds DCR-R, 3b, 3c and 3d are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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