Document [original]

Citation: Thiele, I.; Yehia, H.;

Krausch, N.; Birkholz, M.; Cruz

Bournazou, M.N.; Sitanggang, A.B.;

Kraume, M.; Neubauer, P.; Kurreck,

A. Production of Modified

Nucleosides in a Continuous Enzyme

Membrane Reactor. Int. J. Mol. Sci.

2023,24, 6081. https://doi.org/

10.3390/ijms24076081

Academic Editors: Bettina Siebers

and Salvatore Fusco

Received: 28 February 2023

Revised: 18 March 2023

Accepted: 22 March 2023

Published: 23 March 2023

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

International Journal of

Molecular Sciences

Article

Production of Modified Nucleosides in a Continuous Enzyme

Membrane Reactor

Isabel Thiele 1, Heba Yehia 1,2 , Niels Krausch 1, Mario Birkholz 3, Mariano Nicolas Cruz Bournazou 1,4 ,

Azis Boing Sitanggang 5, Matthias Kraume 6, Peter Neubauer 1and Anke Kurreck 1,7,*

1Department of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin,

Ackerstr. 71-76, ACK24, 13355 Berlin, Germany

2Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research

Institute, National Research Centre, Dokki, Cairo 12622, Egypt

3IHP—Leibniz-Institut für Innovative Mikroelektronik, Im Technologiepark 25,

15236 Frankfurt (Oder), Germany

4DataHow AG, Hagenholzstrasse.111, 8050 Zurich, Switzerland

Department of Food Science and Technology, IPB University, Kampus IPB Darmaga, Bogor 16680, Indonesia

6Department of Chemical and Process Engineering, Technische Universität Berlin, Straße des 17. Juni 135,

10623 Berlin, Germany

7BioNukleo GmbH, Ackerstr. 76, 13355 Berlin, Germany

*Correspondence: [email protected]

Abstract:

Nucleoside analogues are important compounds for the treatment of viral infections or

cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods,

the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continu-

ous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they

offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the

enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and

the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability

of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation

reactions. Over a period of 55 days, 2

-deoxyadenosine was produced continuously, with a product

yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2

-deoxyribonucleoside and

6-chloro-2-fluoro-2

-deoxyribonucleoside were also produced, with high conversion, but for shorter

operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities

comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic

unit was produced, for 2

-deoxyadenosine, 2,6-dichloropurine-2

-deoxyribonucleoside, and 6-chloro-

2-fluoro-2

-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved

enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the

overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

Keywords:

continuous enzyme membrane reactor (EMR); halogenated nucleosides; nucleoside

analogues

; PID controller; purine nucleoside phosphorylase (PNP); pyrimidine nucleoside

phosphorylase (PyNP); Raspberry Pi; thermophilic enzyme; transglycosylation

1. Introduction

Nucleoside analogues are an important class of drugs. They have been widely used

as anticancer, antibacterial, and antiviral medications [

]. The importance of this class of

drugs is also evident in the current COVID-19 pandemic, as the nucleoside analogues mol-

nupiravir and remdesivir were approved for the treatment of SARS-CoV-2 infections [

and many more are currently under investigation in clinical trials [

]. Chemical synthesis is

still the standard procedure for producing nucleoside analogues. The drawbacks, however,

include numerous laborious reaction steps or the formation of various by-products, due to

a lack of stereo- and regioselectivity, which necessitates the implementation of protection

Int. J. Mol. Sci. 2023,24, 6081. https://doi.org/10.3390/ijms24076081 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 6081 2 of 13

and deprotection steps [

]. Hence, often only low product yields are obtained, even

after process optimization [

–

]. In contrast, chemo-enzymatic synthesis routes offer a

suitable alternative, as biocatalysts are active in water-based reaction media and show high

selectivity. Therefore, fewer process steps are required for both synthesis and purification,

and the need for solvents is significantly reduced. Thus, major contributors to the E-factor

(environmental factor, ratio of the mass of waste per mass of product) are minimized,

compared to a wide range of chemical synthesis routes [11].

Nucleoside phosphorylases (NPs) are widely applied for the synthesis of modified

nucleosides [

]. Based on their substrate spectrum, NPs are classified into purine nucleoside

phosphorylases (PNP, EC 2.4.2.1) and pyrimidine nucleoside phosphorylases (PyNP, EC

2.4.2.2). Both catalyze the reversible phosphorolysis of nucleosides to nucleobases and

-D-

pentofuranose-1-phosphates. Transglycosylation reactions are the most common approach

to produce nucleoside analogues, which involve the interchange of a glycosyl moiety be-

tween a sugar donor (nucleoside) and a sugar acceptor (nucleobase) (Figure 1) [

]. In

recent years, thermostable NPs have become increasingly interesting biocatalysts for nucle-

oside synthesis, as reactions at higher temperatures result in increased reaction rates and

improve the solubility of substrates. Additionally, thermostable NPs have been reported to

tolerate high solvent concentrations [

]. Hence, the use of higher substrate concentrations

has led to increased volumetric yields, reduced E-factor values, and facilitated large scale

application [1,11,14].

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 14

molnupiravir and remdesivir were approved for the treatment of SARS-CoV-2 infections

[2,3], and many more are currently under investigation in clinical trials [4]. Chemical syn-

thesis is still the standard procedure for producing nucleoside analogues. The drawbacks,

however, include numerous laborious reaction steps or the formation of various by-prod-

ucts, due to a lack of stereo- and regioselectivity, which necessitates the implementation

of protection and deprotection steps [5,6]. Hence, often only low product yields are ob-

tained, even after process optimization [7–10]. In contrast, chemo-enzymatic synthesis

routes offer a suitable alternative, as biocatalysts are active in water-based reaction media

and show high selectivity. Therefore, fewer process steps are required for both synthesis

and purification, and the need for solvents is significantly reduced. Thus, major contribu-

tors to the E-factor (environmental factor, ratio of the mass of waste per mass of product)

are minimized, compared to a wide range of chemical synthesis routes [11].

Nucleoside phosphorylases (NPs) are widely applied for the synthesis of modified

nucleosides [1]. Based on their substrate spectrum, NPs are classified into purine nucleo-

side phosphorylases (PNP, EC 2.4.2.1) and pyrimidine nucleoside phosphorylases (PyNP,

EC 2.4.2.2). Both catalyze the reversible phosphorolysis of nucleosides to nucleobases and

α-D-pentofuranose-1-phosphates. Transglycosylation reactions are the most common ap-

proach to produce nucleoside analogues, which involve the interchange of a glycosyl moi-

ety between a sugar donor (nucleoside) and a sugar acceptor (nucleobase) (Figure 1)

[12,13]. In recent years, thermostable NPs have become increasingly interesting biocata-

lysts for nucleoside synthesis, as reactions at higher temperatures result in increased re-

action rates and improve the solubility of substrates. Additionally, thermostable NPs have

been reported to tolerate high solvent concentrations [14]. Hence, the use of higher sub-

strate concentrations has led to increased volumetric yields, reduced E-factor values, and

facilitated large scale application [1,11,14].

Figure 1. Enzymatic synthesis of modified nucleosides by nucleoside phosphorylase-catalyzed

transglycosylation. For the synthesis of purine nucleosides, it is advantageous to use pyrimidine

nucleosides as donors, as they are cheaply available and thermodynamically enhance purine nucle-

oside synthesis. In this bi-enzymatic reaction, the PyNP is needed to cleave the donor nucleoside

and the PNP catalyzes the target nucleoside synthesis.

Providing the enzymes for enzymatic reactions on a large scale, is one of the most

cost-intensive factors. Therefore, different methods have been developed that allow the

reuse of active enzymes, or produce valuable products, in continuous processes. Enzyme

immobilization has been most commonly used, and classically, enzymes have been bound

to inert carrier materials [15–21]. Its successful application has already been demonstrated

for nucleoside phosphorylases in several studies, for the synthesis of various natural and

modified nucleosides [18,22,23]. Due to disadvantages of the classical immobilization

techniques, including the use of expensive resins and large losses of enzyme activity due

to immobilization, alternative methods have been developed in recent years. For example,

covalent but reversible immobilization of a purine nucleoside phosphorylase on agarose

microbeads, allowed the recycling of the resin after the enzyme became inactive [24]. In

another approach, crosslinking-based self-immobilization of Escherichia coli uridine phos-

phorylase, even eliminated the need for an external carrier [25].

Figure 1.

Enzymatic synthesis of modified nucleosides by nucleoside phosphorylase-catalyzed

transglycosylation. For the synthesis of purine nucleosides, it is advantageous to use pyrimidine nu-

cleosides as donors, as they are cheaply available and thermodynamically enhance purine nucleoside

synthesis. In this bi-enzymatic reaction, the PyNP is needed to cleave the donor nucleoside and the

PNP catalyzes the target nucleoside synthesis.

Providing the enzymes for enzymatic reactions on a large scale, is one of the most

cost-intensive factors. Therefore, different methods have been developed that allow the

reuse of active enzymes, or produce valuable products, in continuous processes. Enzyme

immobilization has been most commonly used, and classically, enzymes have been bound

to inert carrier materials [

–

]. Its successful application has already been demonstrated

for nucleoside phosphorylases in several studies, for the synthesis of various natural and

modified nucleosides [

]. Due to disadvantages of the classical immobilization

techniques, including the use of expensive resins and large losses of enzyme activity due to

immobilization, alternative methods have been developed in recent years. For example,

covalent but reversible immobilization of a purine nucleoside phosphorylase on agarose

microbeads, allowed the recycling of the resin after the enzyme became inactive [

In another approach, crosslinking-based self-immobilization of Escherichia coli uridine

phosphorylase, even eliminated the need for an external carrier [25].

Despite the progress made, enzyme immobilization still leads to a significant loss of

enzyme activity and to reduced productivity, caused by a limited mass transfer [

Enzyme membrane reactors (EMRs), using freely dissolved enzymes, offer a suitable

alternative, as they combine the advantages of enzyme immobilization (e.g., enzymes

are used until inactivation, easy separation of product and enzyme) and freely dissolved

Int. J. Mol. Sci. 2023,24, 6081 3 of 13

enzymes (e.g., maximum specific activity, no mass transfer limitations). In EMRs, enzymes

are retained in the reactor by membranes with proper molecular weight cut-off (MWCO).

EMRs are scalable and have already been applied to a large variety of products [28–30].

In this study, we tested an EMR system for the continuous synthesis of nucleoside

analogues, using nucleoside phosphorylases of thermophilic origin as biocatalysts. We

adjusted a previously described EMR system, that was successfully used for lactulose

synthesis [

]. As it has been increasingly recognized that microelectronics can provide

valuable technology modules for biotechnology [

], in this work the reactors were operated

with an inexpensive and easy-to-handle proportional-integral-derivative controller (PID-

controller), using a Raspberry Pi computer. The system was established and optimized

for the enzymatic synthesis of deoxyadenosine. Afterwards, the reaction conditions were

adjusted for the efficient synthesis of halogenated nucleoside analogues, as they serve

as a valuable starting material for the preparation of a broad spectrum of nucleoside

analogues [12,13,33].

2. Results and Discussion

2.1. Setup of the EMR

EMRs are widely used for the synthesis of a variety of compounds, in either single or

cascade processes [

], but so far, no reports are available involving natural or modified

nucleosides. Therefore, an automated system of two parallel EMRs was established for

the synthesis of nucleosides, using a feedback controller. The membrane had an MWCO

of <10 kDa, to retain the enzymes in the EMR. To achieve an operation at constant flux,

and thereby a constant residence time, higher pressure is needed to compensate membrane

fouling. The empirically determined PID parameters to achieve a constant flux were

= 6,

= 1.6, and

= 3 (Figure 2B). After less than five minutes from the start of the EMR,

accurate control of the permeate flux was achieved, with only minimal overshooting in

the beginning (Figure 2B). In the further course of the EMR operation, stable control was

achieved over long reaction periods.

2.2. Validation of the Functionality of the EMR Using 20-Deoxyadenosine

To prove the functionality of the EMR controlled by the Raspberry Pi computer, the

synthesis of 2

-deoxyadenosine (dAdo), in a transglycosylation reaction, was studied, using

purified NPs. In this proof-of-concept study, products were not purified, and product

yields were calculated from conversion percentages and volumes of product solutions.

To estimate a suitable hydraulic residence time (HRT), batch reactions were performed

at a 5 mL scale, over a period of 6 h. The one-pot bi-enzymatic reaction reached its

thermodynamic equilibrium after 1 h, with dAdo formation and thymidine cleavage of

95% and 43%, respectively. In the thermodynamic equilibrium, the rates of thymidine

(Thd) cleavage and dAdo formation were equal, and no apparent change in concentrations

were observed anymore. Based on these results, it can be assumed that thermodynamic

equilibrium can be reached at an HRT of 1 h, even in continuous mode. However, initial

experiments showed that a minimum residence time of 4 h was required, because the setup

was not designed to withstand the pressures generated at the required flow rates. Thus,

an operational minimum of 4 h HRT was chosen for the initial dAdo synthesis, to avoid

system leakage.

After setting up the EMR, by loading the substrates and enzymes, the reaction was

started and run in batch mode for 1–2 h. In the initial batch phase, thymidine concentration

decreased over time, while dAdo formation increased simultaneously (Figure S1). After

equilibrium was reached, the formation of dAdo (95%) and cleavage of Thd (40%) was

comparable to batch reactions at the 5 mL scale. Subsequently, continuous mode was started,

with an HRT of 4 h, and dAdo formation and Thd cleavage remained constant compared

to the batch mode. Thus, this residence time was sufficient to reach thermodynamic

equilibrium for the formation of dAdo in the initial phase of EMR operation.

Int. J. Mol. Sci. 2023,24, 6081 4 of 13

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 14

Figure 2. (A) Schematic representation of the experimental setup of a single enzyme membrane re-

actor. During the experiments, two enzyme membrane reactors were run in parallel (C). 1—Com-

pressed air. 2—Substrate tank. 3—Magnetic stirrer. 4—Magnetic drives. 5—Enzyme reactor. 6—Ul-

trafiltration membrane. 7—Heating device. 8—Temperature sensor. 9—Sampling port. 10—Preci-

sion balance. 11—PID controller. (B) PID controller adjustment: screenshots of the tuning of the

parameters to 𝐾



= 6, 𝐾



= 1.6, 𝐾



= 3 and correlation between the target value and measured value

over time. Solid line—target value, dotted line—measured value. Tuning parameters were added

subsequently from the left to the right panel. (C) Photo of the experimental setup of two parallel

reactors during operation.

2.2. Validation of the Functionality of the EMR using 2′-Deoxyadenosine

To prove the functionality of the EMR controlled by the Raspberry Pi computer, the

synthesis of 2′-deoxyadenosine (dAdo), in a transglycosylation reaction, was studied, us-

ing purified NPs. In this proof-of-concept study, products were not purified, and product

yields were calculated from conversion percentages and volumes of product solutions. To

estimate a suitable hydraulic residence time (HRT), batch reactions were performed at a

Figure 2.

(

) Schematic representation of the experimental setup of a single enzyme membrane reactor.

During the experiments, two enzyme membrane reactors were run in parallel (

). 1—Compressed air.

2—Substrate tank. 3—Magnetic stirrer. 4—Magnetic drives. 5—Enzyme reactor. 6—Ultrafiltration

membrane. 7—Heating device. 8—Temperature sensor. 9—Sampling port. 10—Precision balance.

11—PID controller. (

) PID controller adjustment: screenshots of the tuning of the parameters to

= 6,

= 1.6,

= 3 and correlation between the target value and measured value over time.

Solid line—target value, dotted line—measured value. Tuning parameters were added subsequently

from the left to the right panel. (

) Photo of the experimental setup of two parallel reactors during

operation.

dAdo was successfully produced over a period of 55 days. Comparable longevity of

nucleoside phosphorylases has previously been shown for immobilized Halomonas elongata

PNP applied in a flow reactor [

]. With inosine and 6-O-methylguanine as substrates,

the immobilized biocatalyst still showed significant activity after two months of usage for

reactions in flow. Despite the longevity of the enzyme, a loss in activity over time was

observed. After two months of application, the loss in activity was around 50%. A decrease

in activity was also observed in the EMRs. Therefore, HRT had to be regularly adjusted,

Int. J. Mol. Sci. 2023,24, 6081 5 of 13

to ensure steady state conditions and dAdo formation at conversions >90%. Hence, after

22 days and 45 days, the HRT was increased to 6 h and 12 h, respectively (Figure 3A).

Based on this procedure, a productivity of 2.5 g L

−1

was reached for the EMR, which

was in very good agreement with the 5 mL batch reactions. As the EMR was running for

more than 50 days without the need for fresh enzymes, the continuous reaction resulted in

220 times more product per enzymatic unit than in the batch reaction (Table 1).

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 14

Figure 3. Continuous production of 2′-deoxyadenosine from thymidine (25 mM) and adenine (10

mM) using purified nucleoside phosphorylases (150 U of PyNP 02, 1050 U of PNP 02, by BioNukleo)

at 40 °C, stirred at 200 rpm. (A) Monitoring of thymidine (Thd) cleavage and 2′-deoxyadenosine

(dAdo) formation, and regulating pressure during operation, at hydraulic residence times (HRT) of

4, 6, and 12 h. Membrane exchange and centrifugation of the reactor content, to remove precipitate,

is indicated by an arrow. (B) Turbidity of the reaction mixture inside the EMR and membrane clog-

ging were observed over the course of the reaction.

During the operation of the EMR, an increasing turbidity of the reactor content was

observed (Figure 3B). The system pressure rose simultaneously, and critical values were

reached (up to 4 bar). To ensure a continuous EMR performance, the process was stopped

after 6, 14, and 23 days, to change the reactor membrane and to remove the formed pre-

cipitate by centrifugation (Figure 3A). SDS-PAGE analysis revealed that the precipitate

mainly contained the applied biocatalysts (Figure S2). To ensure that solubility of the re-

actants did not lead to precipitate formation, a sample of the precipitate was also analyzed

by high-performance liquid chromatography (HPLC). No peaks corresponding to the nu-

cleosides or nucleobases in the reaction were observed. Denaturation of the biocatalysts,

however, had only a minor eﬀect on dAdo formation in the beginning. After three cycles

of precipitate removal and membrane exchange, however, residence time was prolonged,

to retain dAdo formation >90%.

Figure 3.

Continuous production of 2

-deoxyadenosine from thymidine (25 mM) and adenine (10 mM)

using purified nucleoside phosphorylases (150 U of PyNP 02, 1050 U of PNP 02, by BioNukleo) at

◦

C, stirred at 200 rpm. (

) Monitoring of thymidine (Thd) cleavage and 2

-deoxyadenosine (dAdo)

formation, and regulating pressure during operation, at hydraulic residence times (HRT) of 4, 6,

and 12 h. Membrane exchange and centrifugation of the reactor content, to remove precipitate, is

indicated by an arrow. (

) Turbidity of the reaction mixture inside the EMR and membrane clogging

were observed over the course of the reaction.

During the operation of the EMR, an increasing turbidity of the reactor content was

observed (Figure 3B). The system pressure rose simultaneously, and critical values were

reached (up to 4 bar). To ensure a continuous EMR performance, the process was stopped

after 6, 14, and 23 days, to change the reactor membrane and to remove the formed

precipitate by centrifugation (Figure 3A). SDS-PAGE analysis revealed that the precipitate

mainly contained the applied biocatalysts (Figure S2). To ensure that solubility of the

reactants did not lead to precipitate formation, a sample of the precipitate was also analyzed

by high-performance liquid chromatography (HPLC). No peaks corresponding to the

nucleosides or nucleobases in the reaction were observed. Denaturation of the biocatalysts,

however, had only a minor effect on dAdo formation in the beginning. After three cycles of

precipitate removal and membrane exchange, however, residence time was prolonged, to

retain dAdo formation >90%.

Int. J. Mol. Sci. 2023,24, 6081 6 of 13

Table 1.

Process parameters determined for the synthesis of dAdo and dihalogenated nucleosides.

Parameters were calculated for steady state flow conditions in the EMRs. The threshold value of

conversion was 55% substrate conversion for the calculation of process parameters for EMRs applied,

to produce 6C2FP-dR and 2,6DCP-dR (see Figure 4).

Enzymes Product Productivity

[g L−1h−1]

Specific

Productivity [mg

U−1h−1]

Product Per

Enzymatic Unit

[mg U−1]

Reaction

System Reference

PyNP 02/

PNP 02 dAdo 2.49 0.22 1.32 Batch This study

PyNP 02/

PNP 02 dAdo 2.5 0.22 290.4 * EMR This study

PyNP 02/

PNP 02 2,6DCP-dR 1.6 0.14 0.84 Batch This study

PyNP 02/

PNP 02 6C2FP-dR 1.4 0.12 0.72 Batch This study

PyNP 02/

PNP 02 2,6DCP-dR 1.5

1.6 0.13

0.14 7.8 (HRT 4 h)

33.6 (HRT 8 h/16 h) EMR This study

PyNP 02/

PNP 02 6C2FP-dR 1.3

1.3 0.11

0.11 5.5 (HRT 4 h)

6.49 (HRT 8 h) EMR This study

TtPyNP a/

GtPNP b2,6DCP-R 1.5 - - Batch, immob.

enzyme [13]

TtPyNP/

GtPNP 6C2FP-R 2.0 - - Batch, immob.

enzyme [13]

PNP of Thermus thermophilus,

PyNP of Geobacillus thermoglucosidasius. * During the EMR run, HRT was regularly

adjusted to reach product formation > 90%.

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 14

Table 1. Process parameters determined for the synthesis of dAdo and dihalogenated nucleosides.

Parameters were calculated for steady state flow conditions in the EMRs. The threshold value of

conversion was 55% substrate conversion for the calculation of process parameters for EMRs ap-

plied, to produce 6C2FP-dR and 2,6DCP-dR (see Figure 4).

Enzymes Product

Productivity

[g L

−1

]

Specific

Productivity

[mg U

−1

]

Product Per Enzy-

matic Unit [mg U

−1

]

Reaction

System Reference

PyNP 02/

PNP 02 dAdo 2.49 0.22 1.32 Batch This study

PyNP 02/

PNP 02 dAdo 2.5 0.22 290.4 * EMR This study

PyNP 02/

PNP 02 2,6DCP-dR 1.6 0.14 0.84 Batch This study

PyNP 02/

PNP 02 6C2FP-dR 1.4 0.12 0.72 Batch This study

PyNP 02/

PNP 02 2,6DCP-dR 1.5

1.6

0.13

0.14

7.8 (HRT 4 h)

33.6 (HRT 8 h/16 h) EMR This study

PyNP 02/

PNP 02 6C2FP-dR 1.3

1.3

0.11

5.5 (HRT 4 h)

6.49 (HRT 8 h) EMR This study

TtPyNP

GtPNP

2,6DCP-R 1.5 - - Batch, immob.

enzyme [13]

TtPyNP/

GtPNP 6C2FP-R 2.0 - - Batch, immob.

enzyme [13]

PNP of Thermus thermophilus,

PyNP of Geobacillus thermoglucosidasius. * During the EMR run, HRT

was regularly adjusted to reach product formation > 90%.

Figure 4. Continuous production of modified nucleosides from thymidine (25 mM) and 10 mM 6-

chloro-2-fluoro-purine (A,C), or 2,6-dichloropurine (B,D), using purified pyrimidine and purine nu-

cleoside phosphorylases (150 U of PyNP 02, 1050 U of PNP 02, by BioNukleo GmbH) at 40 °C, stirred

Figure 4.

Continuous production of modified nucleosides from thymidine (25 mM) and 10 mM

6-chloro-2-fluoro-purine (

), or 2,6-dichloropurine (

), using purified pyrimidine and purine

nucleoside phosphorylases (150 U of PyNP 02, 1050 U of PNP 02, by BioNukleo GmbH) at 40

◦

stirred at 200 rpm. Thymidine (Thd) cleavage and formation of the respective nucleoside at residence

times of 4 and 8 h were monitored (

). The threshold value of conversion for the calculation of

process parameters (Table 1) is indicated by the shaded area. Full data sets for the synthesis of the

dihalogenated nucleosides are shown in Figure S4.

Int. J. Mol. Sci. 2023,24, 6081 7 of 13

In batch reactions, the formation of by-products was not observed, using thymi-

dine and adenine as substrates. However, in the EMR, by-products started to form after

2 days of operation. A comparison with reference standards revealed the formation of

-deoxyinosine (dIno) and hypoxanthine (Hx), which are deamination products of dAdo

and adenine (Ade), respectively. By-product concentrations increased with operation time

and longer residence times, reaching a total of up to 5 mM final concentration (50% of

the final product) at the end of the experiment (Figure S3). Deamination is a well-known

challenge in enzymatic reactions and is mainly caused by enzyme impurities, originating

from the heterologous expression host. The application of reaction temperatures above

◦

C [

] or adding deaminase inhibitors like 2

-deoxycoformycin, are suitable options

to overcome this challenge [36].

2.3. Synthesis of Dihalogenated Nucleoside by Nucleoside Phosphorylases

Asthe EMRwas runningwithgood dAdo productivity, the synthesis of 2,6-dihalogenated

nucleoside analogues was performed. To determine a suitable HRT for EMR operation,

batch reactions to form 2,6-dichloropurine-2

-deoxyribonucleoside (2,6DCP-dR) and 6-

chloro-2-fluoropurine-2

-deoxyribonucleoside (6C2FP-dR) were performed initially. As

was also observed for the synthesis of dAdo, the reaction equilibrium was reached after only

1 h. Product conversions were 84% and 83% for 2,6DCP-dR and 6C2FP-dR, respectively, at

equilibrium.

In the batch phase of the EMR runs, equilibrium was also reached within 1 h, with

comparable conversion rates, of 86% and 84% for 2,6DCP-dR and 6C2FP-dR, respectively.

Although an HRT of 4 h was used, due to the operational limitations of the EMR, 6C2FP-dR

formation immediately decreased to 75% and remained constant for 6 h. Thereafter, the

6C2FP-dR formation ranged between 60 and 75%. The formation of 2,6DCP-dR did not

show such fluctuations. Over a period of about 10 h, the 2,6DCP-dR formation was about

80%. Subsequently, it slowly started to decrease. After 50 h, product formation and Thd

cleavage rates strongly decreased for both 2,6DCP-dR and 6C2FP-dR, probably caused

by the inactivation of the biocatalysts. Hence, in comparison to the EMR run for dAdo

synthesis, drastically reduced operation times were observed for the modified nucleosides.

As HRT might be a critical factor for EMR operation, a prolonged HRT of 8 h was studied.

Indeed, for both substrates, operation times increased with prolonged residence times.

A significant decrease in product formation was only observed after 230 h and 62 h, for

2,6DCP-dR and 6C2FP-dR, respectively.

Productivities for small-scale batch reactions, and the EMR at an HRT of 8 h, were

comparable, with values ranging between 1.3 and 1.6 g L

−1

(Table 1). The values

were in good accordance with results observed for the related compounds 2,6-dichloro-

(ß-D-ribofuranosyl)purine (2,6DCP-R) and 6-chloro-2-fluoro-9-(ß-D-ribofuranosyl)purine

(6C2FP-R) using immobilized NPs in batch reactions. Productivities of 1.5 and

2.0 g L−1h−1

were observed for the synthesis of the two ribosides [15].

Product yields per employed enzymatic unit again increased remarkably in the EMRs,

compared to the batch reactions. In the EMRs, with an HRT of 8 h, the product yields per

enzymatic unit were 9 and 40 times higher, for 6C2FP-dR and 2,6DCP-dR, respectively

(Table 1). As stated before, prolonged HRT had a positive effect on product yields for both

dihalogenated products. However, the effect was much more pronounced for 2,6DCP-dR,

where the product yields per employed enzymatic unit increased by a factor of 4.5, when

increasing the HRT from 4 h to 8 h (Table 1).

The lifetime of the enzymes was found to be strongly dependent on the applied

substrates. While the EMR to produce dAdo ran over a period of 55 days, operation

times were much shorter for 6C2FP-dR and 2,6DCP-dR. Halogen substituents have previ-

ously been shown to interfere with enzyme activity or cell viability [

]. Halogenated

nucleoside analogues such as 2-amino-6-chloro-7-deazapurine 2

-deoxyriboside and 6-

amino-2-chloro-7-deazapurine 2

-deoxyriboside, were described as being PNP inhibitors,

by forming a ternary dead-end PNP/base/P

complex, while also being substrates in the

Int. J. Mol. Sci. 2023,24, 6081 8 of 13

reverse synthetic reaction [

]. Furthermore, nucleoside 2

-deoxyribosyltransferase, with a

-deoxy-2

-fluoroglycoside substrate, was found to form a destabilized oxocarbenium-like

transition state, due to the electron-withdrawing fluorine atom at the sugar’s 2

position,

thus leading to accumulation of the covalently bound intermediate and thereby inactivating

the enzyme [

]. Additionally, tissue or organ toxicity has been observed before for halo-

genated nucleoside analogues, such as 2-fluoroadenosine or fludarabine, in pharmaceutical

applications [

–

]. Substitutions with reactive halogens at the purine ring, exert electron

withdrawing centers, that can change the substrate orientation, affinity, pKa, stability, and

impact adjacent groups [45].

3. Materials and Methods

3.1. Reactor Configurations

Two parallel EMRs were used to produce natural and modified nucleosides. The

reactors were set up similarly to the description in Sitanggang et al. [

]. An EMR consists

of a pressure-stable glass container and a holder, which was modified from a XFUF-

047 dead-end test cell (Merck Millipore, Darmstadt, Germany). The maximum working

volume was 92 mL. Flat sheet PES membranes, with a MWCO of 10 kDa (Microdyn

Nadir, Wiesbaden, Germany), were placed at the bottom of the reactor. PTFE tubing (inner

diameter (ID) = 0.8 mm) was employed to connect the substrate tank, enzyme reactor, and

beakers, where the product solution was collected. The EMR was operated in continuous

mode and samples were taken regularly, from a valve located between the enzyme reactor

and product beaker. For permeate measurement, Kern precision balances (Kern & Sohn

GmbH, Balingen-Frommern, Germany) were used. The proportional pressure regulator

was purchased from Festo (MPPE-3-1/4-6-010-B, Esslingen am Neckar, Germany).

Instead of using the Laboratory Virtual Instrument Engineering Workbench (Lab-

VIEW

™

(National Instruments, US), as described by Sitanggang and colleagues, a PID

controller (proportional-integral-derivative controller) was implemented in Python (version

2.7, Python Software Foundation, US), using a Raspberry Pi 3 B (Raspberry Pi Founda-

tion, UK), with the PiXtend V2 -S- extension board, supplied by Qube Solutions GmbH

(Germany).

The weight of the permeate passing through the membrane was measured on a

precision balance, to calculate the deviation from the set hydraulic residence time:

HRT [h]=VR

V(1)

where V

(L) is the reactor volume, and

(L h

−1

) the volumetric flow rate of the feeding

solution. HRT describes the average duration of time that the reactants remain in the EMRs.

Process data (pressure, time, weight, target value) were simultaneously stored in a csv

file and later used to calculate residence times and productivities.

3.2. Control Design

To control the continuous process and ensure a steady conversion rate, a PID controller

was developed, using a Raspberry Pi and the programming language Python. The controller

measures the weight of the harvest tank with the collected product every six seconds and

calculates the deviation (or error I) from the predefined target value, corresponding to the

desired residence time. The output for the valve was calculated as follows:

Out =KPe(t)+KIZe(t)dt +KD

dt e(t)(2)

where

e(t)

is the deviation of the weight from the setpoint at the time point

, and

and

are the respective tuning parameters, reflecting a proportional, an integral, and a

derivative term of the PID controller. The output was then converted, to adjust the pressure

from 0–4 bar, to a value between 0 and 1023, the allowed range of the input for the valve,

Int. J. Mol. Sci. 2023,24, 6081 9 of 13

and sent to the valve via a serial interface. The process time, the target value, as well as the

deviation and the set pressure, were repeatedly (every 6 s) saved to a csv file.

The pressure was not allowed to exceed 4 bar, to ensure the integrity of the device.

Therefore, the membrane was changed if needed, to continue operation within the accept-

able pressure range. In case of insufficient conversion of the substrates, the setpoint of the

residence time was manually increased and set accordingly.

The respective Python script is stored at the following git repository and freely avail-

able: https://git.tu-berlin.de/bvt-htbd/public/thiele_2023_ijms (uploaded on 9 January

2023).

3.3. Chemicals

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), Carbosynth (Compton, Berkshire, UK),

or VWR (Darmstadt, Germany). High-performance liquid chromatography (HPLC) analy-

ses were carried out with an Agilent 1200 series system, equipped with an Agilent diode

array detector (DAD), using a Phenomenex (Torrance, CA, USA) reversed-phase C18 col-

umn (150

4.6 mm). Thermostable nucleoside phosphorylases PyNP Y02 (E-NP-1002) and

PNP N02 (E-NP-2002) in purified form (immobilized metal ion-affinity chromatography),

were provided by BioNukleo GmbH (Berlin, Germany) and used as recommended by the

manufacturer.

3.4. Synthesis of Natural and Modified Nucleosides in Batch Reactions

The products 2

-deoxyadenosine (dAdo), 2,6-dichloropurine deoxyribonucleoside (2,6-

DCP-dR), and 6-chloro-2-fluoropurine deoxyribonucleoside (6C2FP-dR) were synthesized

in a one-pot transglycosylation reaction, using pyrimidine nucleoside phosphorylase PyNP

02 and purine nucleoside phosphorylase PNP 02 as biocatalysts. Thymidine was used

as a sugar donor and adenine, 2,6-dichloropurine, and 6-chloro-2-fluoropurine as sugar

acceptors. In a final volume of 5 mL, a reaction mixture of 25 mM Thd, 10 mM of sugar

acceptor, 1.6 U mL

−1

of PyNP 02, and 11.4 U mL

−1

of PNP 02, in 2 mM potassium phosphate

(KP) buffer (pH 7.0), was prepared. The reaction was incubated at 40

◦

C for 6 h. Regular

samples were taken and analyzed by HPLC-DAD.

3.5. Operational Procedure to Produce Natural and Modified Nucleosides in Enzyme

Membrane Reactors

To perform transglycosylation reactions, substrate concentrations of 25 mM Thd and

10 mM sugar acceptor (adenine, 2,6-dichloropurine, or 6-chloro-2-fluoropurine) were used,

in both the initial batch solution and the feed. The substrates were dissolved in 2 mM KP

buffer (pH 7.0). The syntheses were carried out using 150 U of PyNP 02 and 1050 U PNP

02, at a temperature of 40 ◦C. The agitation speed was 200 rpm.

After reactor assembly, stirring was started and the reactors were filled with substrate

solution, up to 50 mL. Enzymes were added and the reactors were filled up to 92 mL with

substrate solution. Reactions were run in batch mode until equilibrium was reached, after

about 1.5 h. Thereafter, the continuous production of nucleosides and their analogues

was performed with residence times of 4 h. If the system pressure reached almost 4 bar,

the reactor membrane was exchanged, and the reactor content was centrifuged. The

clear supernatant was transferred back into the reaction chamber. To maintain adequate

conversion, HRT was increased up to 16 h, if necessary, during the process.

The productivity of the batch and continuous processes was compared in terms of

specific productivity, productivity/space-time-yield, and product yield per U of enzyme,

and was calculated as follows:

Specific productivity hmg U−1h−1i=cproduct ×Vpermeate

U×∆t(3)

Int. J. Mol. Sci. 2023,24, 6081 10 of 13

with

cproduct

(mg L

−1

) being the product concentration,

Vpermeate

(L) being the permeate

volume, U being the enzyme units, and

∆

t(h) being the reaction period. For batch reactions,

the reaction volume was 5 mL.

Productivity was calculated according to the equation:

Productivity hg L−1h−1i=∑t2

t1P(t)

∑t2

t1V(t)×∆t(4)

where

∑t2

t1P(t)

and

∑t2

t1V(t)

are the amount of product and the permeate volume collected

during a certain period t2−t1, and ∆tthe reaction period.

The enzyme specific product yield was calculated as follows:

Product yield per enzyme unit hg U−1i=∑tend

tstart P(t)

U(5)

where

∑tend

tstart P(t)

is the total amount of product collected over the process time, and U the

enzyme units in the reaction mixture.

3.6. Measurement of Enzyme Activity

All reactions were performed at 2 mL scale, in Eppendorf tubes, which were incubated

in a thermomixer (Eppendorf, Hamburg, Germany) at 40

◦

C and 300 rpm. The standard

activity assay (phosphorolysis) was carried out in potassium phosphate buffer (50 mM; pH

7.0) containing 1 mM uridine or guanosine. After 2 min preheating at the corresponding

temperature, 0.0003 mg mL

−1

(final concentration) of purified enzyme was added to the

mixture, and the reaction was stopped by the addition of

vol. methanol after defined

time intervals, so that <10% of the substrate was converted to the product. Under these

conditions, the reaction rate was linear as a function of time and enzyme concentration.

After centrifugation (20,000

g, 4

◦

C, 15 min), the samples were analyzed by HPLC-DAD.

One unit (U) of enzyme activity was defined as the amount of enzyme catalyzing the

conversion of 1 µmol of substrate per minute, under the respective assay conditions.

3.7. Determination of Nucleosides and Nucleobases by HPLC

HPLC-DAD analyses were performed to monitor the enzyme-catalyzed reactions. For

the synthesis of dAdo, 2,6DCP-dR, and 6C2FP-dR, HPLC analysis was performed with

the following gradient: from 97%, 20 mM ammonium acetate and 3% acetonitrile, to 72%

20 mM ammonium acetate and 28% acetonitrile, in 11 min. The conversion percentages

were determined by measuring the nucleosides and nucleobases at 260 nm (Equation (5)).

Substrates and products (including by-products) were identified by their retention times

and specific spectra. Retention times were determined using pure compounds as standards.

Under these conditions they were as follows: Thd (5.0 min), thymine (Thy) (4.2 min),

dAdo (5.4 min), adenine (Ade) (4.4 min), 26DCP-dR (9.0 min), 2,6-dichloropurine (2,6DCP)

(7.8 min), 6C2FP-dR (8.4 min), 6-chloro-2-fluoropurine (6C2FP) (7.1 min), hypoxanthine (Hx)

(3.2 min), and 2

deoxy-inosine (dIno) (4.5 min). Reaction intermediates, such as phosphate

and deoxyribose-1-phosphate, were not determined, as they are not freely available in

the reaction, since transglycosylation reactions are a balanced interchange of the sugar

moiety between the donor nucleoside and the acceptor. Hence, 2

-deoxyribose-1-phosphate

released after donor nucleoside cleavage by PyNP, is immediately used by the PNP to form

the nucleoside of interest. The inorganic phosphate released in the PNP-catalyzed reaction,

is directly used for donor nucleoside cleavage.

To calculate conversion percentages, first, measured peak areas were converted into

concentrations, based on standard curves obtained from HPLC measurements using de-

Int. J. Mol. Sci. 2023,24, 6081 11 of 13

fined concentrations of the pure compounds (substrates and products). Afterwards, con-

version percentages were calculated, according to:

Conversion [%]=cproduct [mM]

cacceptor [mM]+cproduct [mM]

×100 (6)

where

cproduct

is the concentration of the formed nucleoside, and

cacceptor

is the concentration

of the residual sugar accepting nucleobase. Figure S5 illustrates the calculation of the

conversion using example chromatograms.

4. Conclusions

One of the major challenges for the development of industrial enzymatic processes, is

the economic application of biocatalysts. Within the past decades, enzyme immobilization

techniques have been widely applied, however, enzyme immobilization is often accom-

panied by a loss of activity. Here, we show that EMRs are a suitable alternative for the

synthesis of natural and modified nucleosides, as they facilitated an efficient application of

biocatalysts until their complete loss of activity. Due to the superior stability of thermostable

enzymes, in the present study, the EMRs could run for up to seven weeks, depending on

the applied substrate. Compared to the batch reactions, product yields per enzyme unit

were significantly increased, by factors of 9, 40, and 220 for 6C2FP-dR, 2,6DCP-dR, and

dAdo, respectively, after using prolonged residence times in the EMRs.

Supplementary Materials:

The following supporting information can be downloaded at: https://

www.mdpi.com/article/10.3390/ijms24076081/s1.

Author Contributions:

Conceptualization, A.K., P.N., M.B., I.T., H.Y., A.B.S. and M.K.; methodology,

A.K., P.N., M.B., I.T., H.Y. and A.B.S.; software, I.T. and N.K.; investigation, I.T. and H.Y.; resources,

A.K., M.K., M.N.C.B., M.B. and P.N.; data curation, I.T.; writing—original draft preparation, I.T. and

H.Y.; writing—review and editing, all authors; visualization, I.T.; supervision, M.B., A.K. and P.N.;

funding acquisition, A.K. and P.N. All authors have read and agreed to the published version of the

manuscript.

Funding: H.Y. was funded by the DAAD through the GERLS program.

Data Availability Statement:

Detailed process data can be found in the Supplementary Material.

The Python code is freely accessible via the following git repository: https://git.tu-berlin.de/bvt-

htbd/public/thiele_2023_ijms (uploaded on 9 January 2023).

Acknowledgments:

The authors thank Gabriele Görig-Hedicke and Robert Giessmann for their

help with setting up the enzyme reactors and verifying the program controlling the enzyme reactor,

respectively. The authors are very thankful to Erik Wade for proofreading the manuscript and critical

comments. The authors thank Felix Kaspar for fruitful discussions and Daniel Mika for his advice

regarding electronics components, and the students Fritz Wegner and Minh Chau Luong Boi for

their help during the continuous experiments. We are very grateful that a grant was provided by the

publication fund of the TU Berlin.

Conflicts of Interest:

A.K. is CEO of the biotech company BioNukleo GmbH and P.N. is a member of

the advisory board.

Abbreviations

2,6DCP-dR—2,6-dichloropurine-2

-deoxyribonucleoside, 6C2FP-dR—6-chloro-2-fluoropurine-

-deoxyribonucleoside, Ade—adenine, dAdo—2

-deoxyadenosine, dIno—deoxyinosine, EMR—

enzyme membrane reactor, HPLC—high-performance liquid chromatography, HRT—hydraulic

residence time, Hx—hypoxanthine, ID—inner diameter, MWCO—molecular weight cut-off, NP—

nucleoside phosphorylase, PID controller—proportional-integral-derivative controller, PNP—purine

nucleoside phosphorylase, PyNP—pyrimidine nucleoside phosphorylase, Thd—thymidine.

Int. J. Mol. Sci. 2023,24, 6081 12 of 13

References

Yehia, H.; Kamel, S.; Paulick, K.; Neubauer, P.; Wagner, A. Substrate spectra of nucleoside phosphorylases and their potential in

the production of pharmaceutically active compounds. Curr. Pharm. Des. 2017,23, 13–35. [CrossRef] [PubMed]

Schultz, D.C.; Johnson, R.M.; Ayyanathan, K.; Miller, J.; Whig, K.; Kamalia, B.; Dittmar, M.; Weston, S.; Hammond, H.L.; Dillen,

C.; et al. Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2. Nature

2022

,604, 134–140. [CrossRef]

Scarabel, L.; Guardascione, M.; Dal Bo, M.; Toffoli, G. Pharmacological strategies to prevent SARS-CoV-2 infection and treat the

early phases of COVID-19. Int. J. Infect. Dis. 2021,104, 441–451. [CrossRef] [PubMed]

Ashour, N.A.; Abo Elmaaty, A.; Sarhan, A.A.; Elkaeed, E.B.; Moussa, A.M.; Erfan, I.A.; Al-Karmalawy, A.A. A Systematic Review

of the Global Intervention for SARS-CoV-2 Combating: From Drugs Repurposing to Molnupiravir Approval. Drug Des. Dev. Ther.

2022,16, 685–715. [CrossRef] [PubMed]

5. Mikhailopulo, I.A.; Miroshnikov, A.I. New Trends in Nucleoside Biotechnology. Acta Nat. 2010,2, 36–58. [CrossRef]

Mikhailopulo, I.A.; Miroshnikov, A.I. Some recent findings in the biotechnology of biologically important nucleosides. In

Biochemistry and Biotechnology for Modern Medicine; Komisarenko, S., Ed.; Publishing House Moskalenko O.M.: Kiev, Ukraine,

2013; pp. 328–353.

Ashcroft, C.P.; Dessi, Y.; Entwistle, D.A.; Hesmondhalgh, L.C.; Longstaff, A.; Smith, J.D. Route selection and process development

of a multikilogram route to the inhaled A 2a agonist UK-432,097. Org. Process Res. Dev. 2012,16, 470–483. [CrossRef]

Nguyen-Trung, N.Q.; Botta, O.; Terenzi, S.; Strazewski, P. A Practical Route to 3‘-Amino-3‘-deoxyadenosine Derivatives and

Puromycin Analogues. J. Org. Chem. 2003,68, 2038–2041. [CrossRef]

Divakar, K.J.; Sawant, C.M.; Mulla, Y.A.; Zemse, D.V.; Sitabkhan, S.M.; Ross, B.S.; Sanghvi, Y.S. Commercial-scale synthesis of

protected 20-deoxycytidine and cytidine nucleosides. Nucleosides Nucleotides Nucleic Acids 2003,22, 1321–1325. [CrossRef]

10.

Mackman, R.L. Anti-HIV nucleoside phosphonate GS-9148 and its prodrug GS-9131: Scale up of a 2

-F modified cyclic nucleoside

phosphonate and synthesis of selected amidate prodrugs. Curr. Protoc. Nucleic Acid Chem. 2014,56, 14.10.1–14.10.21. [CrossRef]

11.

Kaspar, F.; Stone MR, L.; Neubauer, P.; Kurreck, A. Route efficiency assessment and review of the synthesis of

-nucleosides: Via

N -glycosylation of nucleobases. Green Chem. 2021,23, 37–50. [CrossRef]

12.

Del Arco, J.; Fernández-Lucas, J. Purine and pyrimidine salvage pathway in thermophiles: A valuable source of biocatalysts for

the industrial production of nucleic acid derivatives. Appl. Microbiol. Biotechnol. 2018,102, 7805–7820. [CrossRef] [PubMed]

13.

Zhou, X.; Szeker, K.; Jiao, L.Y.; Oestreich, M.; Mikhailopulo, I.A.; Neubauer, P. Synthesis of 2,6-dihalogenated purine nucleosides

by thermostable nucleoside phosphorylases. Adv. Synth. Catal. 2015,357, 1237–1244. [CrossRef]

14.

Kamel, S.; Thiele, I.; Neubauer, P.; Wagner, A. Thermophilic nucleoside phosphorylases: Their properties, characteristics and

applications. Biochim. Biophys. Acta Proteins Proteom. 2020,1868, 140304. [CrossRef] [PubMed]

15.

Zhou, X.; Mikhailopulo, I.A.; Cruz Bournazou, M.N.; Neubauer, P. Immobilization of thermostable nucleoside phosphorylases on

MagReSyn

epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides. J. Mol. Catal.

B Enzym. 2015,115, 119–127. [CrossRef]

16.

Okuma, H.; Watanabe, E. Flow system for fish freshness determination based on double multi-enzyme reactor electrodes. Biosens.

Bioelectron. 2002,17, 367–372. [CrossRef]

17.

Carsol, M.A.; Mascini, M. Development of a system with enzyme reactors for the determination of fish freshness. Talanta

1998

,47,

335–342. [CrossRef]

18.

Calleri, E.; Ubiali, D.; Serra, I.; Temporini, C.; Cattaneo, G.; Speranza, G.; Morelli, C.F.; Massolini, G. Immobilized purine

nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications. J. Chromatogr. B

2014,968, 79–86. [CrossRef]

19.

Hori, N.; Watanabe, M.; Sunagawa, K.; Uehara, K.; Mikami, Y. Production of 5-methyluridine by immobilized thermostable

purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859. J. Biotechnol.

1991,17, 121–131. [CrossRef]

20.

Calleri, E.; Cattaneo, G.; Rabuffetti, M.; Serra, I.; Bavaro, T.; Massolini, G.; Speranza, G.; Ubiali, D. Flow-Synthesis of Nucleosides

Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction

Control and Product Purification. Adv. Synth. Catal. 2015,357, 2520–2528. [CrossRef]

21.

Cowan, D.A.; Fernandez-Lafuente, R. Enhancing the functional properties of thermophilic enzymes by chemical modification

and immobilization. Enzym. Microb. Technol. 2011,49, 326–346. [CrossRef]

22.

Rinaldi, F.; Fernández-Lucas, J.; de la Fuente, D.; Zheng, C.; Bavaro, T.; Peters, B.; Massolini, G.; Annunziata, F.; Conti, P.; de la

Mata, I.; et al. Immobilized enzyme reactors based on nucleoside phosphorylases and 2

-deoxyribosyltransferase for the in-flow

synthesis of pharmaceutically relevant nucleoside analogues. Bioresour. Technol. 2020,307, 123258. [CrossRef] [PubMed]

23.

Tamborini, L.; Previtali, C.; Annunziata, F.; Bavaro, T.; Terreni, M.; Calleri, E.; Rinaldi, F.; Pinto, A.; Speranza, G.; Ubiali, D.; et al.

An Enzymatic Flow-Based Preparative Route to Vidarabine. Molecules 2020,25, 1223. [CrossRef] [PubMed]

24.

Benítez-Mateos, A.I.; Paradisi, F. Sustainable Flow-Synthesis of (Bulky) Nucleoside Drugs by a Novel and Highly Stable

Nucleoside Phosphorylase Immobilized on Reusable Supports. ChemSusChem 2022,15, e202102030. [CrossRef] [PubMed]

25.

Visser, D.F.; Hennessy, F.; Rashamuse, J.; Pletschke, B.; Brady, D. Stabilization of Escherichia coli uridine phosphorylase by evolution

and immobilization. J. Mol. Catal. B Enzym. 2011,68, 279–285. [CrossRef]

26.

Kim, S.; Jiménez-González, C.; Dale, B.E. Enzymes for pharmaceutical applications-a cradle-to-gate life cycle assessment. Int. J.

Life Cycle Assess. 2009,14, 392–400. [CrossRef]

Int. J. Mol. Sci. 2023,24, 6081 13 of 13

27.

Sitanggang, A.B.; Drews, A.; Kraume, M. Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose

secondary hydrolysis. Bioresour. Technol. 2014,167, 108–115. [CrossRef]

28.

Cao, T.; Pázmándi, M.; Galambos, I.; Kovács, Z. Continuous production of galacto-oligosaccharides by an enzyme membrane

reactor utilizing free enzymes. Membranes 2020,10, 203. [CrossRef]

29.

Rios, G.M.; Belleville, M.P.; Paolucci, D.; Sanchez, J. Progress in enzymatic membrane reactors—A review. J. Membr. Sci.

2004

,242,

189–196. [CrossRef]

30.

Wöltinger, J.; Karau, A.; Leuchtenberger, W.; Drauz, K. Membrane reactors at Degussa. Adv. Biochem. Eng. Biotechnol.

2005

,92,

289–316. [CrossRef]

31.

Sitanggang, A.B.; Drews, A.; Kraume, M. Development of a continuous membrane reactor process for enzyme-catalyzed lactulose

synthesis. Biochem. Eng. J. 2016,109, 65–80. [CrossRef]

32.

Birkholz, M.; Mai, A.; Wenger, C.; Meliani, C.; Scholz, R. Technology modules from micro- and nano-electronics for the life

sciences. WIREs Nanomed. Nanobiotechnology 2016,8, 355–377. [CrossRef] [PubMed]

33.

Zhou, X.; Szeker, K.; Janocha, B.; Böhme, T.; Albrecht, D.; Mikhailopulo, I.A.; Neubauer, P. Recombinant purine nucleoside

phosphorylases from thermophiles: Preparation, properties and activity towards purine and pyrimidine nucleosides. FEBS J.

2013,280, 1475–1490. [CrossRef] [PubMed]

34.

Yuryev, R.; Strompen, S.; Liese, A. Coupled chemo(enzymatic) reactions in continuous flow. Beilstein J. Org. Chem.

2011

,7,

1449–1467. [CrossRef] [PubMed]

35.

Almendros, M.; Gago JV, S.; Carlos, J.B. Thermus thermophilus strains active in purine nucleoside synthesis. Molecules

2009

,14,

1279–1287. [CrossRef] [PubMed]

36.

Padua, R.; Geiger, J.D.; Dambock, S.; Nagy, J.I. 2

-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and

In Vitro Analysis of Specificity, Potency, and Enzyme Recovery. J. Neurochem. 1990,54, 1169–1178. [CrossRef] [PubMed]

37.

Bzowska, A.; Kulikowska, E.; Shugar, D. Purine nucleoside phosphorylases: Properties, functions, and clinical aspects. Pharmacol.

Ther. 2000,88, 349–425. [CrossRef] [PubMed]

38.

Yehia, H.; Westarp, S.; Röhrs, V.; Kaspar, F.; Giessmann, R.T.; Klare HF, T.; Paulick, K.; Neubauer, P.; Wagner, A. Efficient

Biocatalytic Synthesis of Dihalogenated Thermodynamic Calculations. Molecules 2020,25, 934. [CrossRef]

39.

Anand, R.; Kaminski, P.A.; Ealick, S.E. Structures of Purine 2

-Deoxyribosyltransferase, Substrate Complexes, and the Ribosylated

Enzyme Intermediate at 2.0 Å Resolution. Biochemistry 2004,43, 2384–2393. [CrossRef]

40.

Bonate, P.L.; Arthaud, L.; Cantrell, W.R.; Stephenson, K.; Secrist, J.A.; Weitman, S. Discovery and development of clofarabine: A

nucleoside analogue for treating cancer. Nat. Rev. Drug Discov. 2006,5, 855–863. [CrossRef]

41.

Avramis, V.I.; Plunkett, W. 2-Fluoro-ATP: A toxic metabolite of 9-

-D-arabinosyl-2-fluoroadenine. Biochem. Biophys. Res. Commun.

1983,113, 35–43. [CrossRef]

42.

Skipper, H.E.; Montgomery, J.A.; Thomson, J.R.; Schabel FM, J. Structure-activity relationships and cross-resistance observed on

evaluation of a series of purine analogs against experimental neoplasms. Cancer Res. 1959,19, 425–437. [PubMed]

43.

Bennett, L.L.; Vail, M.H.; Chumley, S.; Montgomery, J.A. Activity of adenosine analogs against a cell culture, line resistant to

2-fluoroadenine. Biochem. Pharmacol. 1966,15, 1719–1728. [CrossRef]

44.

de Giuseppe, P.O.; Martins, N.H.; Meza, A.N.; dos Santos, C.R.; Pereira HD, M.; Murakami, M.T. Insights into Phosphate

Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases.

PLoS ONE 2012,7, e44282. [CrossRef] [PubMed]

45.

Liu, P.; Sharon, A.; Chu, C.K. Fluorinated nucleosides: Synthesis and biological implication. J. Fluor. Chem.

2008

,129, 743–766.

[CrossRef] [PubMed]

Disclaimer/Publisher’s Note:

The statements, opinions and data contained in all publications are solely those of the individual

author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to

people or property resulting from any ideas, methods, instructions or products referred to in the content.