An enzymatic flow-based preparative route to vidarabine

Article abstract of DOI:10.3390/molecules25051223

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase fromAeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

Full text of DOI:10.3390/molecules25051223

molecules
Article
An Enzymatic Flow-Based Preparative Route
to Vidarabine
Lucia Tamborini 1,*, Clelia Previtali 1, Francesca Annunziata 1, Teodora Bavaro 2
,
Marco Terreni 2, Enrica Calleri 2, Francesca Rinaldi 2, Andrea Pinto 3 , Giovanna Speranza 4,
Daniela Ubiali 2,
*
and Paola Conti 1
1
Department of Pharmaceutical Sciences, University of Milan, via Mangiagalli 25, 20133 Milano, Italy;
clelia.previtali@unimi.it (C.P.); francesca.annunziata@unimi.it (F.A.); paola.conti@unimi.it (P.C.)
Department of Drug Sciences, University of Pavia, viale Taramelli 12, 27100 Pavia, Italy;
teodora.bavaro@unipv.it (T.B.); marco.terreni@unipv.it (M.T.); enrica.calleri@unipv.it (E.C.);
francesca.rinaldi01@universitadipavia.it (F.R.)
Department of Food, Environmental and Nutritional Sciences, University of Milan, via Celoria 2,
20133 Milano, Italy; andrea.pinto@unimi.it
Department of Chemistry, University of Milan, via Golgi 19, 20133 Milano, Italy;
giovanna.speranza@unimi.it
2
3
4
*
Correspondence: lucia.tamborini@unimi.it (L.T.); daniela.ubiali@unipv.it (D.U.);
Tel.: +39-02-50319367 (L.T.); +39-0382-987889 (D.U.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Antimo Gioiello and Bruno Cerra
Received: 3 February 2020; Accepted: 7 March 2020; Published: 9 March 2020
Abstract: The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A),
catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside
phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions.
Glyoxyl–agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this
preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and
molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated
yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration)
the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of
CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor
for the preparation of other purine nucleosides.
Keywords: flow bioreactor; enzyme immobilization; nucleoside phosphorylase; vidarabine; nucleosides
1. Introduction
The use of biocatalysis in multiple industrial sectors is rapidly expanding due to the several
benefits that it offers over traditional chemo-catalytic methods. Enzyme-catalyzed reactions are
selective, safe, and environmentally friendly, thus meeting the increasing demand of industry for more
efficient and sustainable processes [
are still the most often encountered bottlenecks in biocatalysis, which limit the implementation of
enzymatic processes in industry [ ]. Continuous flow technology is emerging as a key enabling tool for
biocatalytic process intensification [ ]. Flow processing has the potential to accelerate heterogeneous
1,2]. However, low productivity and difficult product downstream
3
4–8
biotransformations due to biocatalyst high local concentration and enhanced mass transfer, making
large-scale production more economically feasible in small equipment, with a substantial decrease in
reaction time and improvement in space–time yield. Moreover, biocatalyst stability can benefit from
an environment where harsh mixing is avoided. A further advantage of flow reactors is that their
configuration can be easily customized to meet the specific requirements of the biotransformation.
Molecules 2020, 25, 1223; doi:10.3390/molecules25051223
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1223
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In this frame, the addition of in-line purification steps can assist the downstream process, also
reducing time and costs associated with this often troublesome step. Methods based on the use
of biotransformations in continuous flow reactors appear to be suitable for the intensification of
industrially relevant processes, such as the synthesis of active pharmaceutical ingredients (APIs) [
Despite the enormous progress in nucleoside chemistry, the obtainment of nucleoside analogues
is still a synthetic challenge [10 11]. Due to the exquisite selectivity of enzymes, the introduction of one
4,9].
,
or more biocatalytic steps in synthetic sequences has been demonstrated to simplify the preparation of
these molecules, by cutting the number of reaction steps and avoiding by-product formation. Process
intensification by flow reactor technology would be desirable to take another step forward in the full
exploitation of enzyme-based reactions for the synthesis of nucleoside analogues.
Enzymes of nucleic acid metabolism, such as nucleoside phosphorylases (NPs, EC 2.4.2), have
been conveniently used as biocatalysts in the synthesis of a number of nucleoside analogues [12
These enzymes catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the
presence of inorganic phosphate (Pi) to generate the nucleobase and -D-(deoxy)ribose-1-phosphate
–16].
α
(phosphorolysis). If a second nucleobase is added to the reaction medium, the formation of a new
nucleoside can result (transglycosylation).
In this context, we focused our attention on the study of the flow biocatalyzed
transglycosylation reaction to obtain nucleoside analogues of pharmaceutical interest, such as
vidarabine (arabinosyladenine, ara-A). To achieve this, we used recombinant uridine phosphorylase
from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila
(AhPNP), an enzymatic twosome that has been successfully applied to the synthesis of both vidarabine
(“one-pot, two-enzyme” transglycosylation) [17] and vidarabine-50-monophosphate, in a batch mode
(“one-pot, three-enzyme” transglycosylation and phosphorylation) [18].
2. Results and Discussion
With the aim to fully exploit the potential of the target biocatalyzed reaction in flow, we carried out a
study on the co-immobilization of AhPNP and CpUP on two hydrophilic supports, i.e., glyoxyl–agarose
(GA) and EziGTM1 (Opal) [19
,20], which involve a different binding chemistry between the enzymes
and the support (Scheme 1). Both carriers were packed into an Omnifit glass column and the
enzymes were then immobilized by flowing a solution of the proteins through it. The obtained
bioreactors, characterized in terms of immobilization yield and recovered activity, were used to perform
the transglycosylation reaction in a continuous-flow mode. Parameters of the transglycosylation
reaction (e.g., substrate concentration, molar ratio, temperature, pressure, residence time) have been
screened for the highest yield and productivity. An array of substrates was tested to demonstrate the
versatility of the synthetic approach and then to prepare the antiviral drug vidarabine on a gram scale.
The manufacturing process was implemented by adding an in-line purification step for vidarabine to
facilitate work-up and product recovery.

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a) Covalent immobilization on glyoxyl-agarose
OH
O
NH2
ii. NaBH4
i.
ii. NaIO4
i.
O
O
N
O
OH
H
b) Immobilization on EziG1(Opal) by coordination bonds
HO
N
O-
R
H
Mn+
His6
O-
H
R
N
HO
Scheme 1. General scheme of the immobilization reactions discussed in the text. Mn+ = metal ion of
the CPG carrier (controlled pore glass) EziGTM1 (Opal) (e.g., Fe3+, Co2+); His6 = six-histidine tag.
2.1. Immobilization on Glyoxyl–Agarose under Continuous Flow
The flow immobilization of each enzyme was first studied separately. CpUP was immobilized
first, reproducing the batch conditions previously reported [21]. To mimic the batch conditions (i.e., 3 h
at room temperature), the buffer containing CpUP (50 mM sodium carbonate pH 10, protein loading 5
mg per gram of carrier) was flowed for 3 h through the column (glass column i.d.: 6.6 mm; length:
100 mm) packed with glyoxyl–agarose (0.68 g, reactor volume: 0.68 mL) using a recirculation system;
this step was followed by the chemical reduction of the newly formed imino bonds with a solution
of NaBH4 recirculating for 30 min. Under these conditions, both yield and recovered activity of the
immobilized protein were consistent with the results obtained in the in batch immobilization (100% and
35%, respectively). The activity of imm-CpUP was determined spectrophotometrically by monitoring
the phosphorolysis of 20-deoxyuridine to uracil, after withdrawing the immobilized enzyme from the
column. Then, the protocol was repeated by decreasing the immobilization time from 3 h to 1 h and
the reduction step from 30 min to 15 min. The immobilized yield aligned with the previous results
(100%), and the recovered activity was retained (34%).
Immobilization of AhPNP resulted to be troublesome. In fact, under the same conditions as for
CpUP, the percentage of immobilized protein was less than a half (40%) and the recovered activity was
only 14%. The immobilization was performed by recirculating a solution of the free enzyme (5 mg
per gram of carrier) in 50 mM sodium carbonate buffer pH 10 containing hypoxanthine (5 mM) and
glycerol (20% v/v). Again, the reduction of the imino bonds was performed with a solution of NaBH4
recirculating for 30 min.
Each immobilized enzyme recovered from the respective column was submitted to a stability
test. Both CpUP and AhPNP were incubated for 24 h under mechanical stirring in 50 mM carbonate
buffer at pH 10. The enzyme activity was then evaluated after 3, 6 and 24 h and, whereas imm-CpUP
resulted to be stable, imm-AhPNP showed a loss of activity (about 85% after 24 h). Furthermore, both
enzymes were incubated separately for 15 min at 95 ◦C in the presence of a denaturing agent, i.e.,
2-mercaptoethanol; the stability of the immobilized enzymes was not affected in this case.
A flow protocol to evaluate the activity of the immobilized enzymes was also developed with the
aim to have a straightforward method for assessing their activity without the need for “un-packing”
the biocatalysts. In both cases, the phosphorolysis of the reference substrate (i.e., inosine for AhPNP
and 20-deoxyuridine for CpUP) was monitored to determine the residual activity upon immobilization.
Therefore, a solution of inosine (20 mM) or a solution of 20-deoxyuridine (20 mM) in 50 mM phosphate

Molecules 2020, 25, 1223
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buffer pH 7.5 was pumped through the respective bioreactor and the exiting solution was collected and
analyzed by HPLC. The percentage of phosphorolysis was calculated based on the depletion of the
nucleoside, by monitoring the formation of the nucleobase (i.e., hypoxanthine and uracil, respectively;
see Materials and Methods, Section 3.1).
Once the in-flow immobilization for each enzyme was evaluated, a protocol for the in-flow
co-immobilization of the two enzymes was investigated. Given the issues concerning AhPNP
immobilization, we decided to immobilize it first, followed by CpUP (protein loading: 7 mg of
AhPNP and 3 mg of CpUP per gram of carrier), under the conditions reported above. From protein
measurements of the exiting flow solution [22], immobilization yields resulted to be almost quantitative
for CpUP and 58% for AhPNP. After chemical reduction, the recovered activity was 40% for CpUP and
14% for AhPNP.
2.2. Transglycosylation Reaction Using Glyoxyl–Agarose-Based CpUP/AhPNP-Bioreactor
The CpUP/AhPNP -bioreactor (reactor volume: 0.68 mL) was tested in the synthesis of adenosine
(7
, Scheme 2) by transglycosylation reaction using uridine (
1) as the sugar donor (4 mM) and adenine
(5
) as the nucleobase (2 mM) in 50 mM phosphate buffer pH = 7.5. These experimental conditions
(i.e., buffer, substrate concentration and ratio, pH) were selected from previously reported studies in
batch [21]. The solution containing the sugar donor and the sugar acceptor were pumped through the
bioreactor pressurized at 20 psi.
Scheme 2. General scheme of the flow biocatalyzed transglycosylation reactions. BPR: backpressure
0
0
0
regulator (20 psi). Legend: Urd2 OH
(1
, uridine), dUrd2 H
(
2
, 20-deoxyuridine), Thd2 H
(
(
3
7
, thymidine),
0
araU2 OH
(4
, arabinofuranosyluracil), Ade (
, 20-deoxyadenosine), Ino2 OH
5
, adenine), Hpx (
, inosine), dIno2 H
6
, hypoxanthine), Ado2 OH
, adenosine),
0
0
(
10, 20-deoxyinosine), araA2 OH
(11
,
0
0
0
dAdo2 H
(8
(
9
arabinosyladenine, vidarabine), Ura (12, uracil), Thy (13, thymine).
Different residence times and substrate concentrations were screened (Table 1). In only 5 min of
residence time, an 85% conversion was achieved, as determined by HPLC (Table 1, entry 1). Then,
to increase the productivity of the process, the concentration of the sugar donor was set to 100 mM,
keeping constant the 2:1 ratio to the sugar acceptor. Interestingly, also at the highest tested concentration
(Table 1, entry 10), an 89% conversion was obtained in only 5 min. However, due to the poor water
solubility of the substrates, the addition of a co-solvent (i.e., DMF) in the range 10–20% v/v was necessary
(see Table 1). DMF was selected as the co-solvent according to the stability studies previously carried
out in the batch mode transglycosylation [17]. An increase of the residence time up to 20 min (Table 1,
entries 3 and 4) did not lead to a higher conversion. Then, using the highest concentration that does
not require any co-solvent (i.e., 16 mM uridine, 8 mM adenine), a further decrease of the residence time
was evaluated. The reaction reached the equilibrium after only 1 min of residence time (Table 1, entry
7). Finally, an increase of the temperature (up to 38 ◦C) and different molar ratios between the sugar

Molecules 2020, 25, 1223
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donor and the sugar acceptor (1:1 and 4:1) were also tested but, in all cases, no beneficial effects were
observed (data not shown).
Table 1. Synthesis of adenosine (
7) by continuous flow transglycosylation reaction catalyzed by
co-immobilized CpUP and AhPNP: screening of the reaction conditions.
Conversion (%) a
Entry
Residence Time (min)
[Urd, 1] (mM)
[Ade, 5] (mM)
1
2
3
4
5
6
7
5
5
10
20
5
2.5
1
5
4
8
8
2
4
4
4
8
8
8
16
20
50
85
86
84
84
86
86
86
88
89
89
8
16
16
16
32
40
100
8 b
9 b
10 c
5
5
a
Determined by HPLC: [product area/(product area + substrate area)]
×
100. Conversions (%) are the average of
b
c
two experiments. DMF (10% v/v) was added as the co-solvent. DMF (20% v/v) was added as the co-solvent.
Experimental conditions: 50 mM phosphate buffer pH 7.5; T = 28 ◦C; P = 20 psi.
The stability of the bioreactor under continuous work was tested in the conditions reported in
Table 1, entry 7 (reactor volume: 0.68 mL; residence time: 1 min; T = 28 ◦C; P = 20 psi; 16 mM uridine
(1); 8 mM adenine (5); flow stream: 50 mM phosphate buffer pH 7.5) and the exiting flow stream
was collected over 5 days (collected volume ~5 L), monitoring the reaction outcome by HPLC every
6 h. HPLC analysis showed that the enzymes retained their activity (the conversion was constant,
approximately 85%) even after 5 days of continuous work. After that time, the flow stream was stopped.
This result demonstrates the excellent stability of the new bioreactor under the applied operational
conditions (Table 2). Continuous flow synthesis of adenosine (7) resulted in a good space time yield
(1.8 g/day) and a biocatalyst productivity of 0.98 mmolproduct/mgenzyme (defined as mmol of product
formed over 24 h per amount of biocatalyst).
Table 2. Synthesis of adenosine (
7) by continuous flow transglycosylation reaction catalyzed by
co-immobilized CpUP and AhPNP: system productivity.
Catalyst Productivity (24 h)
Residence Time
(min)
Space Time Yield
Conversion (%)
(mmolproduct/mgenzyme
)
(g/day)
1
85
1.8
0.98
Conversion (%) was monitored over 5 days by sampling the exiting flow stream every 6 h. Each analysis was
repeated twice.
The versatility of the bioreactor was evaluated using different sugar donors and sugar acceptors
under the conditions set-up for uridine/adenine transglycosylation reaction (Table 3), resulting in
conversions between 40 and 76%.

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Table 3. Synthesis of purine ribo- and 20-deoxyribonucleosides (
transglycosylation reaction catalyzed by co-immobilized CpUP and AhPNP.
7–10) by a continuous flow
Sugar Donor
Sugar Acceptor
Product
Conversion a (%)
1 (Urd)
1 (Urd)
2 (dUrd)
2 (dUrd)
3 (Thd)
3 (Thd)
5 (Ade)
6 (Hpx)
5 (Ade)
6 (Hpx)
5 (Ade)
6 (Hpx)
7 (Ado)
9 (Ino)
8 (dAdo)
10 (dIno)
8 (dAdo)
10 (dIno)
76
45
70
50
73
40
a
Determined by HPLC: [product area/(product area + substrate area)]
×
100. Conversions (%) are the average
of two experiments. Experimental conditions: 50 mM phosphate buffer pH 7.5; [sugar donor] = 16 mM; [sugar
acceptor] = 8 mM; residence time = 1 min; T = 28 ◦C; P = 20 psi.
2.3. Co-Immobilization on EziGTM Carriers in Continuous Flow
EziGTM is a support made of controlled porosity glass particles, which offers excellent flow through
properties due to its interconnecting pore structure and incompressible/non-swelling nature [19,20].
The particles contain chelated Fe(III) for His-tag binding. Enzymes bind the carrier through a
coordination bond. EziGTM1 (Opal), a hydrophilic carrier, was selected for this study. The flow
co-immobilization (glass column i.d.: 10 mm, length: 100 mm; EziGTM1: 950 mg; reactor volume: 3.0 mL)
was almost quantitative (97%) in about 30 min of residence time, resulting in good recovered activities
(22% for CpUP and 51% for AhPNP). Excellent results were obtained in the model transglycosylation
reaction with uridine/adenine, achieving an 84% conversion in only 1 min of residence time.
2.4. Continuous Flow Synthesis of Ara-A (11) and Product Isolation
In order to synthesize ara-A (11), both the glyoxyl–agarose and the EziGTM1-based bioreactors
were tested. A stock solution containing the sugar donor (araU,
4, 16 mM) and the sugar acceptor
(adenine, , 8 mM) was prepared avoiding any use of organic co-solvent, thus increasing the “greenness”
5
of the protocol and simplifying the product recovery. Residence time was modified with the aim to
achieve the highest conversion (Figure 1). Using the glyoxyl–agarose-based bioreactor, 80% conversion
was reached after 4 h of residence time, whereas, using the EziGTM1-based bioreactor, a similar
conversion was obtained in only 80 min.

Molecules 2020, 25, 1223
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A
100
90
80
70
60
50
40
30
20
10
0
0.1200
0.1000
0.0800
0.0600
0.0400
0.0200
0.0000
10
20
80
120
240
Residence time (min)
B
100
90
80
70
60
50
40
30
20
10
0
1.4000
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
10
20
80
120
240
Residence time (min)
Figure 1. Screening of the residence time for the synthesis of ara-A (vidarabine, 11). (A) enzymes
immobilized on glyoxyl–agarose; (B
) enzymes immobilized on EziGTM1. Experimental conditions:
[araU] = 16 mM; [Ade] = 8 mM; 50 mM phosphate buffer pH 7.5; T = 28 ◦C; P = 20 psi. Conversions
(%; blue bars) were determined by HPLC (see Materials and Methods for details). Specific reaction
rates (
µ
mol min−1
g
−1; orange line) were calculated using the equation: r = [P]
×
f/me, where [P] is
the concentration of the product (
immobilized enzyme (g) [23].
µ
mol mL−1), f is the flow rate (mL min−1) and me is the amount of
Then, the stability of the EziGTM1 bioreactor was tested under continuous work, monitoring the
reaction outcome every 2 h by HPLC. The conversion rapidly (after 24 h) dropped to less than 10% and
protein was detected in the exiting flow stream. Therefore, in the present study, glyoxyl–agarose was
selected as carrier and the bioreactor was used for the continuous flow synthesis of ara-A (11). To this
aim, a larger reactor was prepared (glass column i.d.: 15 mm, length: 150 mm; glyoxyl–agarose: 10 g;
reactor volume: 10 mL) and 1 L of the solution containing the sugar donor and the sugar acceptor was
submitted to the biotransformation, setting the residence time at 120 min (67% conversion). The poor
water solubility of araA was exploited for product recovery. In detail, the exiting flow stream was
directly collected into a cooled vessel to favor the product precipitation. Vidarabine was then recovered
by filtration under vacuum, washed with cooled water and dried under vacuum. The reactor was left
working for 8 days and then the product was recovered in 55% isolated yield and very high chemical

Molecules 2020, 25, 1223
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purity (>99%), as determined by HPLC and NMR. Interestingly, even after 8 days of continuous work,
the conversion was still about 65%.
3. Materials and Methods
All reagents were purchased from Merck Sigma-Aldrich (Milan, Italy). Agarose gel 6B-CL
was activated to glyoxyl–agarose as previously reported [24]. TLC analyses were performed using
commercial silica gel 60 F254 aluminum sheets. HPLC analyses were performed using a Waters 1525
Binary HPLC Pump, equipped with a Waters 2489 UV–vis detector (Waters, Milford, MA, USA) and an
Ascenti C18 column (25 cm
×
4 mm, 4 µm particle size). The Bradford assay and CpUP activity assay
were performed using the spectrophotometer Ultrospec 1000 (Pharmacia Biotech, Cologno Monzese,
1
Italy). H-NMR of ara-A (11) in DMSOd6 was recorded on a Varian Gemini 300 MHz spectrometer
(Varian, Palo Alto, CA, USA). The continuous flow reactions were performed using either a R2+/R4
series or an E series flow reactor, commercially available from Vapourtec (Cambridge, UK) equipped
with Omnifit glass columns with one fixed and one adjustable endfits. Recombinant CpUP and AhPNP
were prepared as previously reported [17]. One international unit (IU) corresponds to the amount of
enzyme that transforms 1 µmol of substrate per minute under specific temperature and pH values,
while the specific activity is defined as units of enzyme activity per milligram of protein.
3.1. Analytical Methods
Samples for the standard activity assay of AhPNP (Section 3.6), flow activity assay of AhPNP
(Section 3.7), flow activity assay of CpUP (Section 3.8) and flow transglycosylation reaction (Section 3.9)
were prepared as reported below. Samples were analyzed by HPLC as follows: injection volume:
10
thymine (13) = 6.6 min; thymidine (
20-deoxyadenosine (
) = 26.4 min; uracil (12) = 3.6 min; uridine (
min; 20-deoxyinosine (10) = 10.9 min; inosine (
µ
L; mobile phase: H2O/MeOH 9:1; flow rate: 1.0 mL min−1
) = 13.2 min; adenine ( ) = 8.9 min; adenosine (7
;
λ
: 260 nm. Retention times (tR):
) = 27.8 min;
) = 4.8 min; 20-deoxyuridine (
) = 7.1
) = 8.2 min. The percentage
3
5
8
1
2
9) = 4.7 min; hypoxanthine (6
of conversion was calculated on the basis of the depletion of the sugar acceptor (nucleobase) and
monitoring the formation of the nucleoside product: Conversion [%] = [product area/(product area +
base area)] × 100.
3.2. Immobilization Yields
The immobilization yields (%) were determined as: immobilized protein and activity recovery
as reported by Sheldon et al. [25] according to the following equations: immobilized protein (%):
(immobilized protein/loaded protein)
enzyme/starting activity) × 100.
×
100; activity recovery (%): (observed activity of the immobilized
3.3. Co-Immobilization of CpUP and AhPNP on Glyoxyl–Agarose under Flow Conditions
Glyoxyl–agarose (0.68 g, volume 0.68 mL) was packed into an Omnifit glass column (6.6 mm
mm) and the packed-bed reactor was washed with 50 mM carbonate buffer pH 10. A mixture of glycerol
(20% v/v) and hypoxanthine (5 mM) in 50 mM carbonate buffer pH 10 (4.4 mL) was prepared. In order
×
100
to dissolve hypoxanthine, the suspension was sonicated using a VWR ultrasonic bath (3 × 10 min
,
frequency: 35 Khz). To the obtained solution, 4.8 mg of AhPNP (156 µL) were added. The solution
was flowed through the column at a flow rate of 0.1 mL min−1; the exiting solution was collected and
recirculated through the column for 3 h. The residual protein was determined by Bradford assay [22].
Then, a solution of 2.0 mg of CpUP (250 µL) in 50 mM carbonate buffer pH 10 (3 mL) was prepared and
flowed through the same column at a flow rate of 0.1 mL min−1. The exiting solution was collected and
recirculated for 3 h. The residual protein was determined by Bradford assay [22]. Chemical reduction
of imines was carried out by adding NaBH4 (14 mg) to the recirculating mixture at 0.2 mL min−1 for
30 min. The bioreactor was then washed with water for 30 min at 0.4 mL min−1
.

Molecules 2020, 25, 1223
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3.4. Co-Immobilization of CpUP and AhPNP on EziGTM1 (Opal) under Flow Conditions
EziGTM1 (950 mg, 3.0 mL) was packed into an Omnifit glass column (10 mm
×
100 mm). A solution
of 25 mg of CpUP stock solution (6.16 mg mL−1) and 25 mg of AhPNP stock solution (22.4 mg mL−1) in
20 mM phosphate buffer pH 7.5 and 300 mM NaCl solution was prepared (16.7 mL). The solution was
flowed at 0.1 mL min−1 and recirculated for 2 h. The residual protein was determined by the Bradford
assay [22].
3.5. Standard Activity Assay of CpUP
The activity of soluble CpUP toward 20-deoxyuridine (
reported [21]. Briefly, a solution of 20-deoxyuridine (
) in 50 mM potassium phosphate buffer
pH 7.5 (230 L) and deionized water (645 L) was prepared. After the addition of the soluble (5 L of
2) was determined as previously
2
µ
µ
µ
enzyme preparation diluted 1:10) or immobilized (10 mg) enzyme, the mixture was incubated under
mechanical stirring. At different times (5 and 10 min), samples were withdrawn and 10 M NaOH
(70 µL) was added. The increase in absorbance at 297 nm was then measured.
3.6. Standard Activity Assay of AhPNP
The activity of soluble AhPNP was assessed by measuring the phosphorolysis of inosine (
previously reported [21]. Briefly, to a 5 mM solution of inosine ( ) in 50 mM phosphate buffer pH 7.5,
the soluble enzyme (30 L of enzyme diluted 1:10) was added and the mixture was incubated under
mechanical stirring. At different times (5 and 10 min), 500 L of the solution were taken and mixed
with 500
L of MeOH. The mixture was heated for 5 min at 95 ◦C and then centrifuged (10 min, 13000
9) as
9
µ
µ
µ
rpm). The supernatant was taken and diluted with a solution of H2O/MeOH (9:1), in order to obtain
1 mM solution. The sample was then analyzed by HPLC. For the immobilized AhPNP, the same assay
was performed by using 20 mg of the immobilized biocatalyst under magnetic stirring. At different
times (5 and 10 min), 500 µL of the solution were taken and filtered. The sample was then diluted
with a solution of H2O/MeOH (9:1) in order to obtain a 1 mM solution. The sample was then analyzed
by HPLC.
3.7. Flow Activity Assay of AhPNP
A solution of inosine (9, 5 mL, 20 mM) in 50 mM phosphate buffer pH 7.5 was prepared and
pumped through the column packed with immobilized AhPNP. The outcome of the reaction at different
residence times (i.e., 1, 2.5, 5 and 10 min) was analyzed by HPLC.
3.8. Flow Activity Assay of CpUP
A solution of 20-deoxyuridine (
2, 5 mL, 20 mM) in 50 mM phosphate buffer pH 7.5 was prepared
and pumped through the column packed with immobilized CpUP. The outcome of the reaction at
different residence times (i.e., 1, 2.5, 5 and 10 min) was analyzed by HPLC.
3.9. General Procedure for the Flow Transglycosylation Reaction
A solution of the sugar donor and the sugar acceptor was prepared in 50 mM phosphate buffer pH
7.5. In the case of hypoxanthine (
6
), in order to dissolve it, the suspension was sonicated (3 × 10 min).
The temperature was set at 28 ◦C. The column packed with immobilized CpUP and AhPNP (0.68 g,
0.68 mL) was washed with 50 mM phosphate buffer pH 7.5 at 0.2 mL min−1 for 10 min at atmospheric
pressure. The substrate solution (2.0 mL) was flowed through the bioreactor. Residence times and
concentrations were varied as reported in Table 1. The reaction outcome was monitored by HPLC. A
sample of the exiting flow stream (200
µL) was diluted with a mixture of H2O/MeOH (9:1), in order to
obtain a sample concentration of 0.1 mM, and was used for the analysis.

Molecules 2020, 25, 1223
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3.10. Synthesis of Vidarabine (11)
A solution of arabinofuranosyluracil (4, 16 mM) and adenine (
5
, 8 mM) was prepared in 50 mM
phosphate buffer pH 7.5 (1 L). The column (15 mm
AhPNP (10 g, 10 mL) was washed with 50 mM phosphate buffer pH 7.5 at 1.0 mL min−1 for 30 min.
The substrate solution was flowed into the system at 83
L min−1 (residence time: 120 min), at 28 ◦C
×
150 mm) packed with immobilized CpUP and
µ
and 20 psi for 8 days. The reaction outcome was monitored by HPLC by collecting a sample of the
exiting flow stream every 8 h. The exiting solution was collected into a flask, which was then cooled at
4 ◦C to favor the product precipitation. The solid was filtered under vacuum, washed with cooled
water (50 mL) and dried under vacuum. Vidarabine (11) was isolated in 55% yield as a white solid
1
(1.1 g). H-NMR was in agreement with literature data [17].
4. Conclusions
The combination of biocatalysis and flow chemistry technology is recognized as an efficient tool
for process intensification, as demonstrated by more and more successful examples of industrially
relevant processes.
In this frame, we have set-up a biocatalyzed synthesis for the preparation of the antiviral drug
vidarabine (11), using a bioreactor in flow. The bioreactor was prepared by in-flow co-immobilization
of CpUP and AhPNP on glyoxyl–agarose. The synthesis was scaled-up to gram scale: 1 g of vidarabine
was synthesized and purified in an overall time of 8 days. The system is versatile and, in principle,
transferable to the preparation of other nucleoside analogues. Further scale-up could be achieved by
simply letting the reactor flow for an extended period of time, without modifying, in any way, the
reaction set-up, or by increasing the size of the bioreactor, or again, by working with parallel reactors.
Author Contributions: Conceptualization, L.T., G.S., D.U. and P.C.; methodology, L.T.; investigation, L.T., C.P.,
F.A., T.B., E.C., F.R., A.P., D.U.; resources, M.T., G.S., D.U., P.C.; data curation, L.T.; writing—original draft
preparation, L.T., G.S., D.U. and P.C.; writing—review and editing, L.T., G.S., D.U. and P.C.; supervision, L.T.,
D.U. and P.C.; project administration, M.T. and P.C.; funding acquisition, M.T. and P.C. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by Cariplo Foundation (Italy), call: “Integrated research on industrial
biotechnologies and bioeconomy 2016”, project BIOFLOW, grant number 2016-0731.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of vidarabine 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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