Biotransformation of halogenated nucleosides by immobilized Lactobacillus animalis 2′-N-deoxyribosyltransferase

Article abstract of DOI:10.1016/j.jfluchem.2016.04.012

An immobilized biocatalyst with 2′-N-deoxyribosyltransferase (NDT) activity, Lactobacillus animalis NDT (LaNDT), was developed from cell free extracts. LaNDT was purified, characterized and then immobilized by ionic interaction. Different process parameters were optimized, resulting in an active derivative (2.6 U/g) able to obtain 1.75 mg/g of 5-fluorouracil-2′-deoxyriboside, an antimetabolite known as floxuridine, used in gastrointestinal cancer treatment. Furthermore, immobilized LaNDT was satisfactorily used to obtain at short reaction times other halogenated pyrimidine and purine 2′-deoxynucleosides such as 6-chloropurine-2′-deoxyriboside (4.9 U/g), 6-bromopurine-2′-deoxyriboside (4.3 U/g), 6-chloro-2-fluoropurine-2′-deoxyriboside (5.4 U/g), 5-bromo-2′-deoxyuridine (2.8 U/g) and 5-chloro-2′-deoxyuridine (1.8 U/g) compounds of pharmaceutical interest in antiviral or antitumor treatments. Besides, increasing the biocatalyst amount 8 times per volume unit allowed obtaining a 5-fold improvement in floxuridine biotransformation. The developed biocatalyst proved to be effective for the biosynthesis of a wide spectrum of nucleoside analogues by employing an economical, simple and environmentally friendly methodology.

Full text of DOI:10.1016/j.jfluchem.2016.04.012

Accepted Manuscript
Title: Biotransformation of halogenated nucleosides by
immobilized Lactobacillus animalis
2ꢀ-N-deoxyribosyltransferase
´
´
Author: Claudia N. Britos Marıa Jose Lapponi Valeria A.
Cappa Cintia W. Rivero Jorge A. Trelles
PII:
S0022-1139(16)30100-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jfluchem.2016.04.012
FLUOR 8768
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Revised date:
Accepted date:
23-2-2016
21-4-2016
22-4-2016
´
´
Please cite this article as: Claudia N.Britos, Maria Jose Lapponi, Valeria A.Cappa,
Cintia W.Rivero, Jorge A.Trelles, Biotransformation of halogenated nucleosides by
immobilized Lactobacillus animalis 2ꢀ-N-deoxyribosyltransferase, Journal of Fluorine
Chemistry http://dx.doi.org/10.1016/j.jfluchem.2016.04.012
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Biotransformation of halogenated nucleosides by immobilized Lactobacillus animalis 2´-N-
deoxyribosyltransferase
Claudia N. Britos, María José Lapponi, Valeria A. Cappa, Cintia W. Rivero, Jorge A. Trelles*
Laboratorio de Investigaciones en Biotecnología Sustentable (LIBioS), Universidad Nacional de Quilmes. Roque
Sáenz Peña 352, Bernal (B1868BXD), Argentina.
*Corresponding author. Tel.: +054 1143657100 (ext. 5645); fax: +54 11 43657132.
E-mail address: jtrelles@unq.edu.ar (Jorge A. Trelles)
1

Graphical abstract
An immobilized biocatalyst with 2’-N-deoxyribosyltransferase activity (NDT) was developed from cell free
extracts, resulting in a derivative with an activity of 2.6 U/g for the biosynthesis of the 5-fluorouracil-2´-
deoxyriboside, an antimetabolite known as Floxuridine, used in gastrointestinal cancer treatment. Furthermore,
immobilized NDT was satisfactorily used to obtain other halogenated pyrimidine and purine 2´-deoxynucleosides
of pharmaceutical interest as antiviral or antitumor compounds at short reaction times. Besides, increasing the
biocatalyst amount 8 times per volume unit allowed obtaining a 5-fold improvement in floxuridine biosynthesis.
The developed biocatalyst proved to be effective for the biosynthesis of a wide spectrum of halogenated
nucleosides by employing an economical, simple and environmentally friendly methodology.
2

Highlights




This work describes a fast, simple and economical immobilized biocatalyst
Lactobacillus animalis NDT was successfully stabilized in DEAE-Sepharose
The developed immobilized biocatalyst was able to sinthetize floxuridine
Halogenated nucleoside analogues of clinical interest were also obtained
3

Abstract
An immobilized biocatalyst with 2´-N-deoxyribosyltransferase (NDT) activity, Lactobacillus animalis NDT
(LaNDT), was developed from cell free extracts. LaNDT was purified, characterized and then immobilized by
ionic interaction. Different process parameters were optimized, resulting in an active derivative (2.6 U/g) able to
obtain 1.75 mg/g of 5-fluorouracil-2´-deoxyriboside, an antimetabolite known as Floxuridine, used in
gastrointestinal cancer treatment. Furthermore, immobilized LaNDT was satisfactorily used to obtain at short
reaction times other halogenated pyrimidine and purine 2´-deoxynucleosides such as 6-chloropurine-2´-
deoxyriboside (4.9 U/g), 6-bromopurine-2´-deoxyriboside (4.3 U/g), 6-chloro-2-fluoropurine-2´-deoxyriboside
(5.4 U/g), 5-bromo-2´-deoxyuridine (2.8 U/g) and 5-chloro-2´-deoxyuridine (1.8 U/g) compounds of
pharmaceutical interest in antiviral or antitumor treatments. Besides, increasing the biocatalyst amount 8 times per
volume unit allowed obtaining a 5-fold improvement in floxuridine biotransformation. The developed biocatalyst
proved to be effective for the biosynthesis of a wide spectrum of nucleoside analogues by employing an
economical, simple and environmentally friendly methodology.
Keywords: Green chemistry, floxuridine, DEAE-Sepharose, antiviral agents, antitumor compounds, enzyme
stabilization.
Chemical compounds studied in this article
Floxuridine (PubChem CID: 5790)
1. Introduction
Nucleoside analogues (NAs) belong to a large family of chemically modified nucleosides with great structural
diversity and broad spectrum of biological activity [1]. NAs represent one of the most important classes of
antiviral and antitumor drugs extensively used in clinical applications [2].
These compounds are mainly synthesized by chemical methods that require the use of organic solvents, multiple
reaction steps and the removal of protecting groups, causing unwanted accumulation of racemic mixtures that
affect further purification [3]. In this sense, the use of biocatalysis for the synthesis of these NAs emerged as an
alternative because reactions are regio- and stereoselective and take place in mild conditions. Two types of
4

enzymes have been mainly used to obtain NAs, such as nucleoside phosphorylases (NPs) and 2´-N-
deoxyribosyltransferases (NDTs, EC 2.4.2.6) [1]. NDTs catalyze the cleavage of the N-glycosidic bond of a 2´-
deoxyribonucleoside via a ping-pong bi- bi mechanism and a covalent deoxyribosyl-enzyme intermediate is
generated, which in turn react with a purine or pirimidine as acceptor base (Scheme 1). These enzymes are
classified into two classes according to their substrate specificity: NDT type I (PDT), specific for purine
exchange, and NDT type II (NDT), which catalyzes the transfer between purines and/or pyrimidines. NDTs, in
comparison with nucleoside NPs, have the advantage of catalyzing transglycosylation reactions between purine or
pyrimidine bases and nucleosides in one step instead of two [4], as with NPs the synthesis of purine NAs using
pyrimidine nucleosides as donors and purine bases as acceptors must be done with two types of NPs, one that is
specific for purines (PNPs; EC 2.4.2.1) and another for pyrimidines (PyNPs; EC 2.4.2.2). NDTs have specificity
for 2´-deoxyribonucleosides [5], although it has been demonstrated that NDTs can accept other related sugar
donors as substrates [6]. Interestingly, it has been reported that some members of lactic acid bacteria (LAB)
express NDTs as part of the nucleoside salvage pathway [7-9] .
Scheme 1. Enzymatic synthesis of NAs and catalytic mechanism of NDTs. The nucleophile glutamic acid (Glu)
present in the enzyme active site participates in the formation the covalent intermediate.
Nowadays, the use of enzymes for industrial applications is limited by several factors, such as the high cost of
recombinant enzymes, instability, and availability in small amounts. Protein stabilization by immobilization has
been exploited to enhance enzyme properties such as activity and specificity for their successful utilization in
industrial processes [10]. Immobilization also promotes product recovery and improves biocatalyst reusability.
The NA 5-fluorouracil-2´-deoxyriboside, better known as Floxuridine (FdUrd), is currently employed in the
treatment of colorectal, pancreatic, kidney and stomach cancer, among others [2, 11]. The fluoro group present in
biologically active molecules gives better characteristics to medicinal compounds. The presence of fluorine
atom(s) into a bioactive nucleoside as an isosteric replacement of hydrogen or as an isopolar mimic of hydroxyl
group, frequently leads to a dramatic change in biological activities and becomes an important strategy in the
5

design and discovery of novel drug candidates[12]. Besides, a related compound such as 5-chlorouracil-2´-
deoxyuriboside proved to be useful in cancer treatment, and both of these halogenated analogues have been used
as substrates (o building block) for the design of new prodrugs [13-15].
The aim of this study was to obtain diverse nucleoside analogues with potential antitumor and antiviral activity. In
this work, we developed a novel immobilized biocatalyst (LaNDT) obtained by an easy and economical procedure
that could reduce the cost of eventual scale-up, and was able to biosynthesize nucleoside analogues useful for
current therapeutics (Scheme 2).
Scheme 2. Biotransformation of pyrimidine and purine NAs performed by the developed immobilized L. animalis
NDT (LaNDT).
2. Results and Discussion
2. 1. Preparation of LaNDT
The presence of NDT activity in Lactobacillus animalis ATCC 35046 was previously described by our group [9,
16]. In order to extract the protein, different lysis methods were employed. When both methods were assayed
separately, poor protein yields were obtained. The use of a combined enzymatic lysis-sonication procedure
improved protein extraction yield (1.3–1.6 mg/mL) by more than 4 times without activity loss.
Then, to enhance the activity of crude extracts, enrichment by ionic exchange and molecular exclusion
chromatography was performed. NDT activity was detected in the elution fractions with the highest ionic strength
(500 mM NaCl).
6

2.2. LaNDT immobilization
NDTs are enzymes that act at mild reaction temperatures; previous results with L.animalis whole cells showed
that the highest activity was obtained at 30 ºC [16], which was further confirmed with L.animalis protein extracts
(data not shown), so this was the selected temperature for the rest of the assays [9]. These results were in
agreement with related NDTs [17] and represent an even friendlier temperature than the reported 40 and 45°C for
other biotransformations using NDTs [18, 19] . Also, different pH values were assayed to characterize LaNDT
and the enzyme showed stability between pH 5 and 8, and optimal activity was observed at neutral to slightly
alkaline pH (data not shown).
Different supports were assayed for NDT immobilization. LaNDT derivatives immobilized in IDA-Agarose,
boronate-agarose and EC-EP Sepharose showed no activity (Table 1). The best immobilization yields were
obtained using cationic supports such as DEAE-Sepharose and Q-Agarose. Then, both derivatives were analyzed
for operational stability by performing subsequent FdUrd reactions, Q-Agarose maintained its activity for 24 h,
while DEAE-Sepharose retained its initial activity for more than 64 h (Table 1). Therefore, this derivative was
selected to optimize immobilization parameters.
Table 1. Immobilization of LaNDT in different supports
DEAE-
Sepharose
Boronate-
Agarose
EC-EP
Sepharose
Q-Agarose
87
IDA-Agarose
Immobilization
yielda (%)
93
65
41
65
Specific activityb
(U/g)
0.030
0.27
64
0.027
0.26
NDc
NDc
-
NDc
NDc
-
NDc
NDc
-
Product yieldb
(mM FdUrd)
Operational
Stability (h)
24
a Immobilization conditions: See “Materials and Methods” for experimental details.
b Reaction conditions: 100 mg of immobilized LaNDT in 400 L reaction volume; donor 6 mM dThd, acceptor 2
mM 5FUra, 20 mM Tris-HCl buffer pH 7.0, 30 ºC, 8 h.
c ND, not detectable.
2.3. Optimization of LaNDT immobilization
Numerous immobilization techniques have been developed to achieve enzyme stabilization. Among them,
adsorption is one of the most efficient methods due to its capacity to retain catalytic activity and most importantly,
because of the possibility to reuse the support after the enzyme has been inactivated [20]. It is worth mentioning
that the ionic exchange of enzymes to charged supports represents a simple non-distorting immobilization method
7

that helps maintain the correct assembly of the oligomeric form and therefore, the quaternary structure is not
altered. This kind of matrix also allows the reuse of the support after desorting the protein after its inactivation
[21].
When the interaction between LaNDT and DEAE-Sepharose was assessed at different pH values (4–8), it was
found that acidic pH negatively affected immobilization yields and therefore, enzymatic activity (Fig. 1A),
probably due to changes in the overall charge of the enzyme at acid pH values that affect its interaction with the
cationic support [22].
Additionally, in order to optimize NDT loading, immobilization on DEAE-Sepharose was performed using
increasing protein mass, and 4.8 times enzyme loading was obtained. This biocatalyst showed 2.6-fold more
activity (0.71 mM of conversion) compared to the initial immobilization conditions (Fig. 1B).
Fig. 1. Optimization of immobilized LaNDT. A: DEAE-Sepharose was immobilized at different pH values, and
FdUdr biotransformation was determined. B: Increasing enzyme amounts were added to 50 mg of support, and
loading capacity of DEAE-Sepharose and FdUdr biotransformation were determined. Reaction conditions: 6 mM
dThd and 2 mM 5FUra in Tris-HCl buffer (20 mM, pH 7.0) at 30ºC and 200 rpm shaking speed.
2.4. Biotransformation of nucleoside analogues
LaNDT capacity to hydrolyze nucleosides with different sugar moieties was studied. 2’-deoxyribonucleosides
(dUrd and dThd), uracil 1-β-D-arabinofuranoside (araUra) and 2´,3´-dideoxyuridine (ddUrd) and were partially
hydrolyzed obtaining yields of 22, 19, 8 and 15 % at 1 h reaction, respectively.
Immobilized LaNDT was used to biosynthesize different nucleoside analogues of pharmaceutical interest. LaNDT
is a NDT II-type, since it can catalyze the synthetic reaction of transglycosylation between pyrimidine and purine
bases (Table 2). 5-Halogenated 2’-deoxyribonucleosides are commonly used as anticancer agents [14]. In this
sense, FdUrd exerts its effects by inhibiting thymidylate synthase [12]. Likewise, BrdUrd and CldUrd have also
8

been used with similar therapeutic effects, [23] and are also used for in vivo studies of cancer cell proliferation
[24]. In this work, FdUrd, CldUrd and BrdUrd were obtained at short reaction times, with specific activities
around 3 U/g, higher than reported for other NDTs [18].
Table 2. Biotransformation of 2'-deoxyribonucleosides by immobilized LaNDTa
Activity
(U/g)
2.6
2.8
1.8
4.3
4.9
5.4
Yield
(%)
35
25
6
61
69
51
Product conversion
Donor Acceptor
Product
(mg/g)
1.75
1.54
0.84
3.84
3.76
2.77
dThd
5FUra
FdUrd
5BrUra
5ClUra
6BrPur
6ClPur
6Cl2FPur
BrdUrd
CldUrd
6BrPdR
6ClPdR
6Cl2FPdR
dThd
a Reaction conditions: 10 mg of immobilized LaNDT in 100 L reaction volume: donor 6 mM, acceptor 2 mM, 20
mM Tris-HCl buffer pH 7.0, 30 ºC.
Purine bases modified at the 6-position and their derivatives have received considerable attention, due to their
structural similarity to DNA damage products arising from the modification of N-6 of 2′-deoxyadenosine or O-6
of 2′-deoxyguanosine [25]. These compounds possess a wide range of biological properties. It was shown that
2ChPur or 2FPur nucleosides have anticancer activity [26]. 6-modified purine 2´-deoxyribosides (6ClPdR,
6BrPdR and 6Cl2FPdR) have been obtained with high values of specific activity and yields between 50 and 70 %
using immobilized LaNDT (Table 2). Also, the bioconversion of 6ClPdR and CldUrd nucleosides was achieved
with LaNDT and not with immobilized whole cells as previously reported [9]. This nucleoside can act as building
block to synthesis guanosine derivatives [6] or may be used for the synthesis of modified antisense
oligonucleotides [27].
Many 2´,3´-dideoxynucleosides have been used because of their anti-HIV activity [28]. Immobilized LaNDT
could synthesize 5-fluoro-2´,3´-dideoxyuridine (FddUrd) and 5-fluorouracil-arabinonucleoside (ara5FUra) after
24 and 8 h, respectively (data not shown). Additionally, in view of a potential bioprocess scale-up, increasing the
biocatalyst amount 8 times per volume unit resulted in a 5-fold increase in FdUrd activity (Fig. 2), making
LaNDT a promising biocatalyst for future application in the development of compounds with high added value
and pharmacological interest.
9

Fig. 2. Different derivative masses per volume were added, relative activity was determined. Reaction conditions:
6 mM dThd and 2 mM 5FUra in Tris-HCl buffer (20 mM, pH 7.0) at 30 ºC and 200 rpm shaking speed.
3. Conclusions
In this work a novel biocatalyst from Lactobacillus animalis (LaNDT) was developed, prepared and purified from
cell free extracts. When LaNDT was immobilized in DEAE-Sepharose, it remained associated at high ionic
strength, which allowed purification and immobilization in a single step, simplifying the process and reducing the
costs of derivative preparation. Besides, the immobilized biocatalyst can be recovered after deactivation by
subjecting the support to a new enzyme loading. Optimization of immobilization parameters allowed reaching
1.75 mg/g of FdUrd. Besides, immobilized LaNDT was used to biosynthesize other purine and pyrimidine 2’-
deoxyribonucleosides with yields above 50 % at short reaction times.
On the other hand, the developed biocatalyst showed capacity to accept 2´,3´-dideoxyribose and arabinose
moieties as substrates, obtaining FddUrd and ara5FUra in one step reaction. These results indicate that NDT from
Lactobacillus animalis ATCC 35046 could be used to produce a wide range of nucleoside analogues of
pharmaceutical interest employing an environmentally friendly methodology.
4. Experimental
4.1. Materials
Nucleosides and bases were purchased from Sigma Chem. Co. (Brazil). Culture media compounds were obtained
from Britania S.A. (Argentina). Chemicals were purchased from Sigma Chem. Co. (Brazil). HPLC solvents used
were supplied by Sintorgan S.A. (Argentina). Supports (DEAE-Sepharose, IDA-Agarose, Q-Agarose, Boronate,
10

EC-EP Sepharose, Sephadex G-75) were purchased from Sigma Aldrich (Argentina). Ultrafiltration devices were
acquired from Sartorius (Argentina).
4.2. Growth conditions
Lactobacillus animalis ATCC 35046 was grown according to the required conditions of temperature and culture
medium until saturation, harvested by centrifugation for 10 min at 11000 x g, washed twice with Tris-HCl buffer
(20 mM, pH 7) and stored at -20 ºC until use.
4.3. Enzyme preparation from L. animalis crude extracts
Cell free protein extracts of L. animalis were obtained by subsequent rupture methods: enzymatic lysis and
sonication. First, bacterial suspension was incubated in Tris-HCl buffer (10 mM, pH 8) with 1 mg/mL lysozyme
at 37 ºC and 200 rpm for 2 h. Crude extracts were recovered by centrifugation at 5500x g for 30 min. Then,
sonication was carried out using 5 cycles of 5 min pulses at 8–9 W (Sonic Dismembrator F60, USA), and protein
extract was obtained by centrifugation at 5500x g for 30 min at 4 ºC. Then, LaNDT was recovered and enriched
by ion exchange and then, by gel filtration chromatography. A DEAE-Sepharose column was equilibrated with
Tris-HCl buffer (10 mM, pH 7), and stepwise elution was performed with increasing concentrations of NaCl (0–
500 mM). The fraction with NDT activity was further purified by gel filtration chromatography through a
SephadexTM G-75 column equilibrated with Tris-HCl buffer (10 mM, pH 7). Elution fractions were quantified by
UV absorption at 280 nm and assayed for NDT activity. The protein fractions containing LaNDT were pooled and
concentrated by ultrafiltration (VivaspinTM devices, 10,000 MWCO, Sartorius). The protein extracts were
quantified by the coomassie blue method, and analyzed by polyacrylamide gel electrophoresis (PAGE) and NDT
activity as described below.
4.4. Standard assay for LaNDT
Biotransformation of FdUrd from thymidine (dThd) and 5-fluorouracil (5FUra) was selected as standard reaction
to evaluate enzymatic activity. Free or immobilized LaNDT was added to a solution containing 6 mM dThd and
2mM 5FUra in Tris-HCl buffer (20 mM, pH 7.0) at 30ºC and 200 rpm shaking speed. At different times, 20 L
aliquots were taken, centrifuged at 10,000 g and the supernatant was analyzed by HPLC. In such conditions, one
unit of enzyme (U) was defined as the amount of enzyme that catalyzed the formation of 1 mol of FdUrd in 1
min.
11

4.5. LaNDT immobilization
4.5.1. Immobilization by adsorption
One hundred mg of support (Q-Agarose, DEAE-Sepharose, Boronate-Agarose or IDA-Agarose) was incubated
with 0.27 mg of LaNDT. The immobilization mixture was gently stirred for 4 h at 4 ºC, washed with 20 mM Tris-
HCl buffer (pH 7), dried by vacuum filtration and stored at 4 ºC until use.
4.5.2. Immobilization by covalent bonding
One hundred mg of EC-EP Sepharose was incubated with 0.27 mg of LaNDT in 20 mM Tris-HCl buffer (pH 7)
with 1 M NaCl. The mixture was gently stirred for 8 h at 4 ºC, and the support was separated from the solution by
filtration. The obtained derivative was incubated for 8 h at 4 ºC in 3 M glycine (pH 9) and stored at 4 ºC until use.
In both cases, immobilization yields were determined as the difference in protein content of the mixture before
and after incubation with the supports. NDT activity of the obtained derivatives was evaluated by FdUrd standard
assay.
4.6. Operational stability of immobilized LaNDT
FdUrd biotransformation with immobilized LaNDT on DEAE-Sepharose and Q-Agarose was carried out several
times until inactivation.
4.7. Optimization of LaNDT immobilization
LaNDT was immobilized on DEAE-Sepharose at different pH values (4, 5, 6, 7 and 8) using 2.7 mg of total
protein per gram of support. Once the optimal value was selected, loading capacity was evaluated using 50 mg of
support and increasing amounts of LaNDT in the immobilization mixture. Immobilization yields were determined
by quantification of total protein content, and the activity of the derivatives was evaluated by FdUrd standard
assay.
4. 8. Biotransformation of nucleoside analogues
Thymidine (dThd) and 2´-deoxyuridine (dUrd) were assayed as sugar donors. Different purine and pyrimidine
bases were tested: 5-fluorouracil (5FUra), 5-bromouracil (5BrUra), 5-chlorouracil (5ClUra), 6-chloropurine
(6ClPur), 6-bromopurine (6BrPur) and 6-chloro-2-fluoropurine (6Cl2FPur). Reactions were performed using 100
12

mg/mL of immobilized LaNDT, 6 mM nucleoside and 2 mM base, 30 ºC and 200 rpm. At different times (5-8 h),
20 L aliquots were taken and centrifuged at 10,000x g, and the supernatant was analyzed by HPLC to evaluate
yield expressed as percentage and product conversion expressed as mg of product per gram of support.
4.9. Amount of biocatalyst
In order to maximize the performance of the biocatalyst obtained, FdUrd biotransformation was performed using
different amounts of immobilized LaNDT at constant reaction volume.
4.10. Analytical Methods
Nucleoside analogues were quantitatively measured by HPLC (Gilson) with a Nucleodure 100 C-18 column (5
m, 125 mm × 5 mm) at 254 nm using water/methanol as mobile phase. The ratios of mobile phase and retention
time for the reaction products were : 95:5 (v/v) for 5-fluoruracil-2´-deoxyriboside (FdUrd, 5.8 min); 90:10 (v/v)
for 5-bromouracil-2´-deoxyriboside (BrdUrd, 11 min), 6-chloropurine-2´-deoxyriboside (6ClPdR, 2.9 min), 6-
bromopurine-2´-deoxyriboside (6BrPdR, 2.9 min) and 6-chloro-2-fluoropurine-2´-deoxyriboside (6Cl2FPdR, 6.2
min), 99:1 (v/v) for 5-chlorouracil-2´-deoxyriboside (CldUrd, 8.7 min) and 98:2 (v/v) for 5-fluorouracil-2´,3´-
dideoxyriboside (FddUrd, 7.1 min) and 5-fluorouracil-1-β-D-arabinofuranoside (Ara5FUra, 3.9 min).
Product identification was performed by MS-HPLC LCQ-DECAXP4 Thermo Spectrometer with the electron
spray ionization (ESI) method. Phenomenex C18 column (5 µm, 100 mm × 2 mm) and Xcalibur 1.3 software
(Thermo-Finnigan, USA) were used. Mobile phase and flow were: (i) 95/5 (v/v) water/methanol + 0.1% acetic
acid (F: 200 L/min) for FdUrd (M+: 246.8), CldUrd (M+: 266.7) and BrdUrd (M+: 307.2); and (ii) 15/85 (v/v)
water/methanol + 0.1% acetic acid (F: 200 L/min) for 6ClPdR (M+: 271.8), 6Cl2FPdR (M+: 289.9), 6BrPdR
(M+: 315.8), 5FddUrd (M+: 231.1) and Ara5FUra (M+: 262.9).
Acknowledgments
This research was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2013-2658),
Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2014-KA5-00805) and Universidad Nacional de
Quilmes (PUNQ 1409/15). CWR and JAT are research members at CONICET. MJL and VAC are CONICET
research fellows.
13

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