Production, characterization and synthetic application of a purine nucleoside phosphorylase from Aeromonas hydrophila

Article abstract of DOI:10.1002/adsc.201100505

Purine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over-expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6-substituted purine-9-ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6-oxo- and 6-aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1-, 2-, 6- and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleosides are not. A few transglycosylation reactions were carried out using 7-methylguanosine iodide (4) as a d-ribose donor and 6-substituted purines as acceptor. In particular, following this approach, 2- amino-6-chloropurine-9-riboside (2c), 6-methoxypurine- 9-riboside (2d) and 2-amino-6-(methylthio)purine- 9-riboside (2g) were synthesized in very high yield and purity.

Full text of DOI:10.1002/adsc.201100505

FULL PAPERS
DOI: 10.1002/adsc.201100505
Production, Characterization and Synthetic Application of a
Purine Nucleoside Phosphorylase from Aeromonas hydrophila
a,d
b
a
b
Daniela Ubiali, Carla D. Serra, Immacolata Serra, Carlo F. Morelli,
a,d
c
b,d,
Marco Terreni, Alessandra M. Albertini, Paolo Manitto, *
b,d,e,
and Giovanna Speranza
*
a
Dipartimento di Scienze del Farmaco, Universitꢀ degli Studi di Pavia, via Taramelli 12, I-27100 Pavia, Italy
Dipartimento di Chimica Organica e Industriale, Universitꢀ degli Studi di Milano, via Venezian 21, I-20133 Milano, Italy
Fax : (+39)-02-5031-4072; phone: (+39)-02-5031-4097; e-mail: giovanna.speranza@unimi.it
Dipartimento di Genetica e Microbiologia, Universitꢀ degli Studi di Pavia, via Ferrata 1, I-27100 Pavia, Italy
Italian Biocatalysis Center, via Taramelli 12, I-27100 Pavia, Italy
b
c
d
e
Istituto di Scienze e Tecnologie Molecolari, CNR, via Golgi 19, I-20133 Milano, Italy
Received: June 29, 2011; Published online: January 12, 2012
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100505.
Abstract: Purine nucleoside phosphorylase (PNP) sides are not. A few transglycosylation reactions
from Aeromonas hydrophila encoded by the deoD were carried out using 7-methylguanosine iodide (4)
gene has been over-expressed in Escherichia coli, pu- as a d-ribose donor and 6-substituted purines as ac-
rified, characterized about its substrate specificity ceptor. In particular, following this approach, 2-
and used for the preparative synthesis of some 6-sub- amino-6-chloropurine-9-riboside (2c), 6-methoxypur-
stituted purine-9-ribosides. Substrate specificity to- ine-9-riboside (2d) and 2-amino-6-(methylthio)pur-
wards natural nucleosides showed that this PNP cata- ine-9-riboside (2g) were synthesized in very high
lyzes the phosphorolysis of both 6-oxo- and 6-amino- yield and purity.
purine (deoxy)ribonucleosides. A library of nucleo-
side analogues was synthesized and then submitted
to enzymatic phosphorolysis as well. This assay re- Keywords: Aeromonas hydrophila; biotransforma-
vealed that 1-, 2-, 6- and 7-modified nucleosides are tions; chemoenzymatic synthesis; glycosylation; nu-
accepted as substrates, whereas 8-substituted nucleo- cleoside phosphorylases; nucleosides
Introduction
chemical methods which suffer from low stereoselec-
tivity, multi-step procedures and modest total yields.
[3]
Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze Several procedures based on the “transglycosylation”
the reversible cleavage of the glycosidic bond of (de- reaction (Scheme 1) have been employed (using both
oxy)ribonucleosides in the presence of inorganic or- isolated enzymes and whole cells) to prepare a wide
[
4–11]
thophosphate (P) as a second substrate to generate variety of nucleosides.
i
the nucleobase and a-d-(deoxy)ribose-1-phosphate
Structural analysis revealed that there are two dis-
tinct families of NPs: NPI and NPII. Members of the
NPI family show either a trimeric or a hexameric qua-
ternary structure. Enzymes belonging to this large
family accept a wide range of purine nucleosides
[
1,2]
[
(d)R-1-P)] [Eq. (1)].
(
PNPs) and uridine (UP). Members of the NPII
If a second nucleobase is added to the reaction family display a dimeric structure and are specific for
[
1]
medium the formation of a new nucleoside can result. pyrimidine nucleosides.
Thus, NPs provide a potentially suitable tool for che-
With regard to purine nucleoside phosphorylases
moenzymatic synthesis of both natural and unnatural (PNPs), the most studied member of NPI family,
[
12]
nucleosides. To obtain the b-oriented glycoside bond Bzowska et al.
proposed a classification based on
their use appears to be a convenient alternative to molecular mass (mm), subunit composition and sub-
96
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Adv. Synth. Catal. 2012, 354, 96 – 104

Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase
[7]
Scheme 1. Typical one-pot transglycosylation reaction.
strate specificity. It comprises two major groups of en-
zymes: a low-mm class (80–100 kDa) including homo- tential of A. hydrophila purine nucleoside phosphor-
trimers (mainly from mammalian sources) which rec- ylases (PNPs, E.C. 2.4.2.1) as biocatalysts for practical
These findings prompted us to investigate the po-
AHCTUNGTRENNUNG
ognize inosine and guanosine as natural substrates, uses.
and a high-mm class (110–160 kDa), whose members
In this paper, we describe the cloning and over-ex-
possess a hexameric structure and are specific for ade- pression of one of the A. hydrophila PNPs, the testing
nosine in addition to inosine and guanosine. of its substrate specificity, and a few examples of syn-
Generally speaking, bacteria mostly produce either thetic applications.
homotrimers or homohexamers, while mammalian
PNPs are homotrimeric.
Purine nucleosides bearing different substituents in Results and Discussion
the 6 position are an important class of compounds
frequently endowed with biological activities. Some of Cloning, Expression and Purification of PNP from
them were found to exert cytotoxic, cytostatic, antivi- Aeromonas hydrophila
[
13–17]
ral and antitumor effects.
In addition, a few 6-
substituted purine nucleosides in combination with In the sequenced genome of Aeromonas hydrophila
[
21]
proper PNPs have been exploited in a “suicide gene subsp. hydrophila ATCC 7966 there are three genes
[
12,18–20]
therapy” treatment for solid tumors.
In particu- annotated as PNPs: deoD, deoD1 and xapA. Since
lar, the “gene-directed enzyme/prodrug therapy” em- xapA usually codes for a purine nucleoside phosphor-
ploys a gene encoding an enzyme that is capable of ylase with specificity restricted to 6-oxopurine nucleo-
converting a non-toxic prodrug into a potent cytotoxic sides, for our purposes only the deoD and deoD1
agent. In the above context, because of the difference genes were considered because they are believed to
in substrate specificity between human purine nucleo- code for a PNP with a broader substrate specificity,
[
1]
side phosphorylases (hPNPs) and bacterial PNPs,
accepting both 6-oxo- and 6-aminopurine nucleosides.
the latter (e.g., PNP from E. coli) have been shown to deoD and deoD1 belong to the PNP UDP 1 super
be potential therapeutic agents for cancer treatments family which includes not only PNPs, but also uridine
when used in combination with appropriate prodrugs, phosphorylase (UP) and 5’-(methylthio)adenosine
[
1]
i.e., nucleosides possessing a highly cytotoxic nucleo- phosphorylase (MTAP).
base (e.g., 6-methylpurine-2’-deoxyribose, which does Hypothetic genes coding the desired enzymatic ac-
[
19]
not act as a substrate for the hPNPs but is activated tivity were subjected to attempted amplification from
by E. coli PNP with release of the cytotoxic 6-methyl- the genomic DNA (extracted from the strain reported
[
14]
purine).
by Trelles et al. and kindly donated by Prof. A. M.
[
14]
In 2005, Trelles et al. carried out a screening of Iribarren) by using specific primers designed on the
catalytically active microorganisms for the synthesis basis of the sequence of Aeromonas hydrophila subsp.
of 6-modified (deoxy)ribonucleosides via transglyco- hydrophila ATCC 7966. However, it was only possible
sylation reactions using uridine or thymidine as a to amplify the deoD gene, while attempts to amplify
sugar donor and 6-substituted purines as an acceptor deoD1 did not afford any amplification product even
(
see Scheme 1). Among the tested microorganisms, when degenerated primers were used in the PCR re-
Aeromonas hydrophila appeared to be particularly in- actions. This evidence led us to hypothesize that the
teresting for synthetic purposes due to a considerably deoD1 gene is not present in this Aeromonas hydro-
broad range of specificity.
phila strain, or that its sequence should greatly differ
Adv. Synth. Catal. 2012, 354, 96 – 104
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97

Daniela Ubiali et al.
FULL PAPERS
Table 1. Activity of Ah PNPII with respect to some natural
[a]
purine (deoxy)ribonucleosides.
[b]
Entry
Substrate
Specific activity [IU/mg]
1
2
3
4
5
6
inosine (1a)
2’-deoxyinosine
guanosine (1b)
2’-deoxyguanosine
adenosine (2a)
2’-deoxyadenosine
75
137
26
28
35
28
[c]
[c]
[
a]
Experimental conditions: 50 mM phosphate buffer,
pH 7.5, room temperature, volume=10 mL, [substrate]=
5
mM.
IU=mmol min .
substrate]=1 mM.
[
[
b]
c]
À1
[
from the sequence reported in the sequenced
genome.
The primary sequence of deoD deduced from the
coding sequence obtained by PCR showed 99.6%
identity and 100% similarity compared to the se-
quence reported in the NCBI genome database
(
Aero
ACHTUNGTRENNUNGm onas hydrophila subsp. hydrophila ATCC
7966). In particular, after PCR amplification, only
one conservative substitution was found (I60V) and,
as expected, after insertion into pET/TOPO 151 ex-
pression vector, the resultant coding sequence did not
show any other mutation.
The protein was expressed as 29 kDa fusion protein
carrying an N-terminal His tag in order to facilitate
6
purification by affinity chromatography. After purifi-
cation, more than 90% purity was obtained with a
final concentration in the range 4.0–6.0 mg/mL. deoD-
PNP was then characterized for its substrate specifici-
ty towards certain natural substrates (Table 1). deoD-
Figure 1. Purine ribonucleosides examined as substrates of
PNPs from Aeromonas hydrophila (Ah PNPII) and Bacillus
subtilis (Bs PNPI).
PNP was found to catalyze the phosphorolysis of both (v/v) of glycerol, the enzyme completely maintained
-oxo- and 6-aminopurine (deoxy)ribonucleosides, its activity after 24 h incubation. The residual activity
6
with a strong preference for (deoxy)inosine. Hence- from stability assays was evaluated by HPLC by
forth, also taking into account its hexameric structure measuring the formation of hypoxanthine when the
and the high molecular mass of 155 kDa, we refer to enzyme was incubated with inosine.
[12]
it as Ah PNPII.
The stability of Ah PNPII was assayed at pH 7.5 in
the presence of glycerol (10–20% v/v). In fact, glycer- Substrate Specificity of PNP from Aeromonas
ol is routinely used for enzyme storage due to its sta- hydrophila (Ah PNPII)
bilizing and preservative properties and can be used
in up to 60% concentration without causing protein To evaluate the substrate specificity and the catalytic
[22]
inactivation. Glycerol was therefore the first-choice efficiency of the prepared Ah PNPII, nucleoside ana-
cosolvent to perform the phosphorolysis reactions of logues (1c–j, 2b–f, 3a,b and 4 in Figure 1) were sub-
a collection of purine nucleoside analogues (see mitted to enzymatic phosphorolysis (Table 2).
Figure 1) which were shown to be sparingly soluble in
From an inspection of Table 2 it clearly appears
phosphate buffer. The use of glycerol was accompa- that 1-, 2-, and 7-modified nucleosides are accepted as
nied by a positive effect on the substrate solubility substrates. The equilibrium was shifted toward the nu-
which allowed us to work, for all synthesized sub- cleoside phosphorolysis although to different extents.
2
strates, at 1 mM concentrations.
In fact, in the case of N -methylguanosine (1c), 1-
In the case of Ah PNPII, at room temperature and methylinosine (1d) and 1-methylguanosine (1e),
pH 7.5, in 50 mM phosphate buffer containing 20% about 30–50% of unreacted nucleoside was detected
98
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Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase
[a]
Table 2. Phosphorolysis of purine ribonucleosides by Ah PNPII (see Experimental Section for the standard procedure).
Entry
Compound
Unreacted nucleoside
[b]
at 6 h and 258C [%]
[
c]
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
inosine (1a)
45 (35)[
c]
guanosine (1b)
60 (64)
2
[c]
N -methylguanosine (1c)
32 (100)[
31 (100)[
48 (100)
c]
c]
[
1-methylinosine (1d)
1-methylguanosine (1e)
8-bromoinosine (1f)
8-bromoguanosine (1g)
8-(methylamino)inosine (1h)
8-(ethanolamino)inosine (1i)
8-(n-butylamino)inosine (1j)
adenosine (2a)
6-chloropurine-9-riboside (2b)
2-amino-6-chloropurine-9-riboside (2c)
6-methoxypurine-9-riboside (2d)
2-amino-6-methoxypurine-9-riboside (2e)
6-(methylthio)purine-9-riboside (2f)
6-thioinosine (3a)
c]
c]
c]
c]
c]
100 (100)[
100 (100)
[
100 (100)[
100 (100)[
0
1
2
3
4
5
6
7
8
9
100 (100)
[
c]
c]
c]
c]
c]
52 (100)[
25 (100)
[
59 (100)[
23 (100)[
55 (100)
[c,d)
32 (n.t.)
[c]
50 (23)[
c]
6-thioguanosine (3b)
7-methylguanosine iodide (4)
57 (43)
[c]
0 (0)
[
a]
Experimental conditions: 50 mM phosphate buffer and glycerol 20% (v/v), pH 7.5, room temperature, volume=20 mL,
[substrate]=1 mM.
b]
[
[
[
Estimated by HPLC analysis.
c]
Percentage in brackets refers to Bs PNPI-catalyzed reactions.
n.t.=not tested
d]
at the equilibrium, whereas in the case of 7-methyl- synthesized nucleoside analogues was performed as
guanosine iodide (4) this compound was totally con- previously described for Ah PNPII.
verted into 7-methylguanine. Interestingly, also the
Data of Table 2 reveal marked differences between
tested 6-substituted nucleosides (2b–f and 3a, b) were the two PNPs in their substrate specificity. While the
recognized as substrates. This result appears particu- activity of Bs PNPI appears to be restricted to ino-
larly important due to the biological relevance of sine, guanosine (1a, b), their 6-thio analogues (3a, b)
these analogues and, therefore, in the view of synthe- and 7-methylguanosine iodide (4), all being character-
sizing them through a convenient enzyme-based ap- ized by the lactam-lactim or thione-thiol tautomerism
[
23,24]
proach by transglycosylation. By contrast, all the in the pyrimidine ring,
Ah PNPII extends its ac-
2
tested 8-substituted nucleosides (1f–j) were found to tivity also to 1-, N - and 6-substituted purine ribonu-
be inactive as substrates for Ah PNPII.
cleosides (1c–e and 2a–f).
Analogously to the experimental design followed
Both enzymes were shown to be unable to accept
for Ah PNPII, all modified nucleosides (except for 8-substituted inosines and guanosines (1f–j). In this
2
f) were tested as substrates in the phosphorolysis case, considering that the 8-unsubstituted purine nu-
catalyzed by PNPI from Bacillus subtilis (Bs PNPI).
cleosides exist in aqueous solution predominantly as
[25]
This enzyme, previously produced and character- anti-conformers,
the lack of activity as substrates
[
5,7]
ized by some of the present authors,
Ah PNPII and can be classified as a PNPI. In fact, position can be attributed to strong stereo constraints
its mm is estimated to be ca. 120 kDa and its specifici- forcing the molecule to assume a syn-conforma-
ty is directed to 6-oxopurines; its tetrameric quater- tion
nary structure is consistent with the exception ac-
differs from shown by nucleosides having a bulky substituent in 8-
[
12]
[
7]
[26,27]
which is not recognized by the enzyme.
Other differences in substrate specificity between
counted by Bzowska et al. for Bacilli sp. within the the two PNPs we examined may be interpreted in
[7,12]
low mm family.
terms of different substrate/enzyme interactions in-
Bs PNPI was found to maintain 50% of its activity volved in the catalytic action.
after 10 h in 50 mM phosphate buffer containing 20%
glycerol at room temperature and pH 7.5. The enzy- states (T.S.) have been proposed,
Two substrate binding modes and related transition
[
28,29]
each belonging
matic assay for evaluating Bs PNPI activity as well as to one of the PNP classes (I or II) mentioned before
for monitoring the phosphorolysis reactions with the (see Figure 2). They are supported by crystallographic
[
12,28]
studies and functional analysis.
Adv. Synth. Catal. 2012, 354, 96 – 104
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99

Daniela Ubiali et al.
FULL PAPERS
Figure 2. Proposed catalytic mechanism (transition state structures are featured) for class I (A) and class II (B) purine nu-
[28]
[29,30]
cleoside phosphorylases, e.g., human PNP and E. coli PNP,
respectively.
The 6-oxo (or 6-thio) groups in the purine nucleus 1e and 1c, respectively, and Table 2) can be explained
of substrates accepted by class I enzymes (low mm, as being due to the different environments of the sub-
trimeric or tetrameric) is essential to stabilize the de- strate molecule inside the active site of the two en-
veloping negative charge in the T.S. by hydrogen zymes (see Figure 2). In fact, for class I PNPs the hy-
bonding with an asparagine residue (Asn243 in drogen bond interaction of N-1ÀH with a glutamate
[28]
human PNP).
residue (Glu201 in human PNP) has been suggested
Such a T.S., depicted in Figure 2(A), accounts for to prevent reconstitution of the keto tautomer, thus
the lack of enzymatic activity of Bs PNPI towards 6- favoring the enzymatic process in the phosphorolytic
[
28]
alkyl-, 6-alkylthio-, 6-alkoxy-, 6-amino-, and 6-halo- direction [Figure 2, (A)].
purine ribonucleosides (see Table 2). Therefore, it seems plausible that the presence of a
On the other hand, concerning the broader sub- methyl group linked to N-1 or of a bulky group in the
strate specificity exhibited by class II enzymes (high 2 position, so making the above interaction impossible
mm, hexameric structure, e.g., Ah PNPII), which are or strongly hindered, actually hampers the cleavage
active not only towards inosine and guanosine but of the glycosidic bond from its completion.
also towards adenosine and a number of other 6-sub-
stituted purine nucleosides, there is a general consen- few transglycosylation reactions were carried out
To explore the synthetic potential of Ah PNPII, a
[
29,30]
sus
that the protonation of N-7 in the imidazole using 7-methylguanosine iodide (4) as a d-ribose
ring by an aspartic acid residue (Asp204 in E. coli donor and 6-substituted purines (5) as acceptor
PNP) is the prerequisite for the heterolytic break- (Scheme 2). This procedure takes advantage of both
down of the glycosidic bond [Figure 2, (B)].
the irreversible phosphorolysis of the 7-methyl ribo-
[
31]
It is worthwhile to note that the difference in activi- side (4) to 7-methylguanine (6) and the ready prep-
1
ty between the two PNPs with respect to N -methyl- aration of this nucleoside derivative from commercial-
2
[32]
as well as N -methyl-6-oxopurine nucleosides (i.e., 1d, ly available guanosine. When performed on a prep-
Scheme 2. Synthesis of 6-substituted ribonucleosides 2c, 2d, 2g, 2h via transglycosylation catalyzed by Ah PNPII. Isolated
yields of produced nucleosides are reported. Experimental conditions: 50 mM phosphate buffer, pH 7.5, room temperature,
volume=20 mL, molar ratio 4/5=3:1 (2c), 4:1 (2d, 2g, 2h). n.i.=not isolated; 2h was prepared only on an analytical scale
(
see Experimental Section for details).
100
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Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase
arative scale, 2-amino-6-chloropurine-9-riboside (2c), Prior to analysis, the enzyme was filtered off from the reac-
tion medium by centrifugation through centrifugal filter de-
vices (10 kDa MWCO, VWR International).
6
-methoxypurine-9-riboside (2d) and 2-amino-6-
(
methylthio)purine-9-riboside (2g) were obtained in
Purification of products was accomplished either by flash
chromatography (silica gel 60, 40–63 mm, Merck) or by prep-
arative HPLC using an AKTA Basic100 instrument (Phar-
macia, Uppsala, Sweden); chromatographic conditions were
as follows: column, LiChrosorb RP-18 (7 mm, 250ꢄ25 mm,
Merck); flow rate, 10 mL/min; detector, l=254 nm; mobile
pure form and in high yield (up to 93%, Scheme 2).
Conclusions
The broad substrate specificity exhibited by Ah phase MeOH-H O, isocratic elution at 3% MeOH for
2
PNPII, in particular to catalyze the reversible phos- 20 min, then gradient elution from 3% to 100% MeOH in
phorolysis of 6-chloro-, 6-alkoxy-, 6-alkylthio-, and 6- 45 min.
1
13
H and C NMR spectra were recorded at 400.13 and
alkylpurine nucleosides appears of notable relevance
[
2]
100.61 MHz, respectively, on a Bruker AVANCE 400 spec-
trometer equipped with the XWIN-NMR software package
in view of using this enzyme for synthetic purposes
[
18,19]
and as a tool in gene therapy.
1
13
(
Bruker, Karlsruhe, Germany) at 300 K. H and C chemi-
Because of this activity, Ah PNPII has been exploit-
ed for the regio- and stereoselective synthesis of a
few nucleoside analogues (2c, d, g, h) by a “one-pot,
one-enzyme” transglycosylation. The use of easily
available 7-methylguanosine iodide (4) as the sugar
cal shifts (d) are given in parts per million and were refer-
enced to the solvent signals (d =2.50 and d =39.50 ppm
H
C
13
for DMSO-d ). C NMR signal multiplicities were based on
6
APT (attached proton test) spectra.
Electrospray ionization mass spectra (ESI-MS) were ac-
donor and the very high yield and purity of the pro- quired on a ThermoFinnigan LCQ Advantage spectrometer
duced nucleosides make this chemoenzymatic ap- (Hemel Hempstead, Hertfordshire, U.K.).
proach a valuable, greener alternative to totally chem-
[33]
ical strategies.
Studies on the immobilization of Ah PNPII on
solid supports are now in progress with the aim to re-
cycle the biocatalyst for repeated use.
Cloning, Expression and Purification of Ah PNPII
Bacterial strains: The Aeromonas hydrophila strain was
kindly provided by Prof. A. M. Iribarren (University of
Quilmes, Buenos Aires, Argentina). The bacterial strain E.
coli DH5a was used for the propagation of all the plasmids.
Experimental Section
À
Genotype: F endA1 glnV44 thi-1 recA1 relA1 gyrA96
deoR nupG F80dlacZDM15 D(lacZYA-argF)U169, hsdR17-
General
À
+
A
H
U
G
R
N
U
G
ACHTUNGTRENUNNG( DE3) was
K
PfuUltra DNA polymerase was from Stratagene (Agilent
Technologies, Life Sciences and Chemical Analysis, Wald-
bronn, Germany). pET/TOPO 151 wector was purchased
from Invitrogen (San Giuliano Milanese, Italy), and the re-
employed for the overexpression of all the enzymes. Geno-
type: F ompT gal dcm lon hsdSB
lacUV5-T7 gene 1 ind1 sam7 nin5]).
Extraction of genomic DNA: The A. hydrophila genomic
DNA was extracted from cells grown in NB (Difco) medium
at 308C. Cells collected by centrifugation were resuspended
in 2.5 mL of 50 mM Tris HCl pH 8.0, 5 mM EDTA, 50 mM
NaCl and 5 mg/mL lysozime, incubated for 5 min at room
temperature and then at 558C for 30 min. The lysate was
cleared by addition of 0.4 mg/mL of proteinase K and l% of
SDS and incubation at 558C for 10 min. The DNA purified
by two phenol-chloroform extractions was precipitated from
the aqueous phase with two volumes of absolute ethanol,
collected on a sealed Pasteur pipette tip and dissolved in
0.1 mL of 10 mM Tris HCl pH 8.0 and 1 mM EDTA.
ꢂ
combinant protein was purified by using Protino Ni-TED
1000 packed columns (MACHEREY-NAGEL GmbH & Co.
KG, Dꢃren, Germany).
Inosine (1a), guanosine (1b), adenosine (2a), (and their
’-deoxy counterparts), 6-(methylthio)purine-9-riboside (2f),
-methylpurine-9-riboside (2h), 6-(methylthio)purine, 6-
2
6
methylpurine, 2-amino-6-chloropurine, 6-methoxypurine and
all other reagents were purchased from Sigma–Aldrich
(
Milano, Italy) and/or from VWR International (Milano,
Italy) and were used without further purification. All sol-
vents were of HPLC grade.
Analytical TLC was performed on silica gel F254 precoated
aluminum sheets (0.2 mm layer; Merck, Darmstadt, Germa-
ny); components were detected under an UV lamp (l=
Production of the recombinant protein: The putative NP
genes of A. hydrophila were identified using the Basic Local
[34]
Sequence Alignment Tool (BLAST).
The corresponding
2
54 nm) and by spraying with a ceric sulfate/ammonium mo-
open reading frames were subjected to attempted amplifica-
tion from the genomic DNA by PCR using PfuUltra DNA
polymerase (Stratagene) and primers with overhangs specif-
ic for cloning into the pET/TOPO 151 vector (Invitrogen).
deoD forward: 5’-CACCATGGCAACTCCTCATATCAATG-3’;
deoD reverse: 5’-TTATTAGTTGTCGCCCAGCAG-3’; deoD1
forward: 5’-CACCATGACCGCTCATATCAAT-3’; deoD1 re-
verse: 5’-TTATTACAGCGCGGTGTACAGCG-3’.
lybdate solution, followed by heating to ca. 1508C.
Enzymatic reactions were monitored by analytical HPLC
using a Waters Model 600E liquid chromatographer (Waters
Corp., Milford, MA) connected to an HP1050 diode-array
detector (Hewlett–Packard GmbH, Waldbronn, Germany)
and the following chromatographic conditions: column, Li-
Chrosorb RP-18 (5 mm, 250ꢄ4.6 mm, Merck); flow rate,
1
.0 mL/min; detector, l=254 nm; mobile phase: MeOH-
Only the reaction using the deoD primers gave a PCR
product that was verified by direct sequencing. The deoD
H O, gradient elution from 3% to 100% MeOH in 20 min.
2
Adv. Synth. Catal. 2012, 354, 96 – 104
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
101

Daniela Ubiali et al.
FULL PAPERS
gene was cloned into the pET/TOPO 151 vector and ex-
8-(Methylamino)inosine (1h): Prepared from 2’,3’,5’-tri-O-
[39]
[40]
pressed as fusion protein with an N-terminal His tag.
acetyl-8-bromoinosine
according to Ref.
and purified
6
Cultures of E. coli BL21 ACHUTNRGNENUG( DE3) transformed with the
pET151-AhdeoD were grown in Luria Bertani medium sup-
plemented with 100 mg/mL ampicillin. Cells were grown at
by flash chromatography, eluent: CH Cl /MeOH=7/1; yield:
2
2
1
95%. H NMR (400 MHz, DMSO-d ): d=2.81 (d, J=
6
4.8 Hz, 3H, CH -NH-C8), 3.65 (m, 2H, H-5’), 3.95 (d, J=
3
3
78C until the OD600 reached 0.6–0.8. Then the cultures
2.4 Hz, 1H, H-4’), 4.11 (br s, 1H, H-3’), 4.56 (m, 1H, H-2’),
5.12 (d, J=3.6 Hz, 1H, HO-5’), 5.29 (d, J=6.4 Hz, 1H, HO-
3’), 5.68 (t, J=4.4 Hz, 1H, HO-2’), 5.85 (d, J=7.2 Hz, 1H,
were induced for 3 h with IPTG (isopropyl-b-d-thiogalacto-
pyranoside) to a final concentration of 1 mM. Upon IPTG
addition, the temperature was immediately lowered to 288C.
Cells were harvested by centrifugation at 5000 rpm for
H-1’), 6.81 (q, J=4.8 Hz, 1H, CH -NH-C8), 7.82 (s, 1H, H-
3
1
3
2), 12.08 (br s, 1H, NH-1); C NMR (100 MHz, DMSO-d ):
6
3
0 min and the proteins were extracted by sonication. Cell
debris was harvested by centrifugation at 13000 rpm for
0 min at 48C. More than 90% of the over-expressed Ah
d=29.78 (CH ), 62.21 (C-5’), 71.38 (C-3’), 71.86 (C-2’), 86.42
3
(C-4’), 87.40 (C-1’), 122.64 (C-5), 143.08 (C-2), 148.51 (C-8),
+
1
152.15 (C-4), 156.29 (C-6); ESI-MS: m/z=320.3 [M+Na] ;
PNPII was in the soluble fraction of the crude cell extract.
The enzyme was purified from the supernatant by using
Protino Ni-TED 1000 packed columns (Macherey-Nagel)
HPLC: t =11.2 min.
R
8
-(Ethanolamino)inosine (1i): To a solution of 2’,3’,5’-O-
ꢂ
[39]
triacetyl-8-bromoinosine (100 mg, 0.21 mmol) in dry diox-
ane (1 mL) was added ethanolamine (180 mL, 2.94 mmol)
under an N2 atmosphere, and the mixture was stirred at
following the instructions provided by the supplier. Pellets,
crude extracts, supernatants and collected fractions were an-
alyzed by SDS-PAGE (4–12%) and Comassie-blue staining.
The fractions showing the presence of a band (about 90% of
the total proteins) of the expected size (29 kDa) were
pooled and dialyzed against 50 mM phosphate buffer pH 7.5
and 10% glycerol. The final concentration of the protein
was determined to be in the range 4.0–6.0 mg/mL by Brad-
8
08C while monitoring the reaction progress by TLC
(
CH Cl /MeOH=7/2). After 24 h, the solvent was removed
2
2
under reduced pressure and the residue was purified by
flash chromatography (CH Cl /MeOH=7/3) to give pure 1i;
2
2
1
yield: 53%. H NMR (400 MHz, DMSO-d ): d=3.40 (m,
6
CH -NH-C8), 3.55 (m, 2H, CH -CH -NH-C8), 3.63 (m, 2H,
2
2
2
[35]
fordꢅs method. The yield of Ah PNPII expression in E.
coli was 100–200 mg of 99% pure protein per liter of cul-
ture.
H-5’), 3.96 (m, 1H, H-4’), 4.12 (d, J=3.6 Hz, 1H, H-3’), 4.60
(
(
t, J=6.4 Hz, 1H, H-2’), 5.87 (d, J=7.2 Hz, 1H, H-1’), 6.83
br s, 1H, -NH-C8), 7.81 (s, 1H, H-2); C NMR (100 MHz,
13
DMSO-d ): d=45.41 (-CH -NH-C8), 60.28 (-CH -CH -NH-
6
2
2
2
C8); 61.96 (C-5’), 71.31 (C-3’), 71.91 (C-2’), 86.15 (C-4’),
7.08 (C-1’), 122.03 (C-5), 143.43 (C-2), 148.10 (C-8), 151.21
(C-4), 157.02 (C-6); ESI-MS: m/z=350.2 [M+Na] ; HPLC:
tR =11.9 min.
Ah PNPII Activity Assay
8
+
To a solution of inosine (13.5 mg, 0.05 mmol) in 50 mM
phosphate buffer pH 7.5 (10 mL), Ah PNPII was added
(
100 mL of 1:10 diluted protein solution) and the mixture
8-(n-Butylamino)inosine (1j): Prepared from 2’,3’,5’-tri-O-
acetyl-8-bromoinosine according to Ref.[39] and purified
[39]
was stirred at room temperature. Aliquots (0.2 mL) of the
reaction mixture were withdrawn at fixed time intervals (2,
4
by flash chromatography, eluent: CH Cl /MeOH=7/2;
2
2
1
, 6 and 8 min), filtered by centrifugation (10 kDa MWCO
yield: 75%. H NMR (400 MHz, DMSO-d ): d=0.89 (t, 3H,
6
centrifugal filter devices, 5000 rpm, room temperature,
J=7.3 Hz, CH -CH -CH -CH -NH-C8), 1.35 (m, 2H, CH -
3
2
2
2
3
4
t
min) to remove the protein and analyzed by HPLC;
CH -CH -CH -NH-C8), 1.55 (m, 2H, CH -CH -CH -CH -
NH-C8), 3.27 (m, CH -CH -CH -CH -NH-C8), 3.64 (m, 2H,
3 2 2 2
2 2 2 3 2 2 2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(inosine)=9.2 min; t (hypoxanthine)=7.1 min.
R
R
The enzyme activity (IU/mg) was calculated as follows:
H-5’), 3.96 (m, 1H, H-4’), 4.10 (br s, 1H, H-3’), 4.53 (m, 1H,
H-2’) 5.14 (d, J=4.0 Hz, 1H, HO-5’), 5.28 (d, J=6.4 Hz,
1H, HO-3’), 5.66 (t, J=4.4 Hz, 1H, HO-2’), 5.87 (d, J=
7.6 Hz, 1H, H-1’), 6.80 (t, J=5.2 Hz, 1H, NH-C8), 7.80 (s,
IU/mg={[(substrate concentration (mM)ꢄ% of conver-
sion)/time (min)]ꢄvol (mL)}/mg of protein.
One IU corresponds with an amount of enzyme that con-
verts one mmol of inosine into hypoxanthine per min.
1
3
1H, H-2), 12.02 (br s, 1H, NH-1); C NMR (100 MHz,
DMSO-d ): d=14.64 (CH -CH -CH -CH -NH-C8), 20.53
6
3
2
2
2
(
CH -CH -CH -CH -NH-C8), 31.67 (CH -CH -CH -CH -
3 2 2 2 3 2 2 2
Synthesis of Modified Nucleosides
NH-C8), 42.76 (CH -CH -CH -CH -NH-C8), 62.20 (C-5’),
7
3
2
2
2
2
2
1.22 (C-3’), 71.78 (C-2’), 86.43 (C-4’), 87.24 (C-1’), 122.50
N -Methylguanosine (1c): Prepared from N -methyl-2’,3’-O-
[36]
(
C-5), 142.93 (C-2), 148.41 (C-8), 151.46 (C-4), 156.25 (C-6);
isopropylideneguanosine
in 70% yield by acid-catalyzed
+
cleavage of the isopropylidene group (80% formic acid) fol-
ESI-MS: m/z=362.14 [M+Na] ; HPLC: t
=14.1 min.
R
1
lowed by purification via preparative HPLC. H NMR and
6-Chloropurine-9-riboside (2b): Prepared according to
1
3
[37]
+
[41]
C NMR, see Ref. ; ESI-MS: m/z=298 [M+H] ; HPLC:
tR =12.1 min.
-Methylinosine (1d): Prepared according to Ref.
Ref.
CH
ESI-MS, see Ref. ; HPLC: t
2-Amino-6-chloropurine-9-riboside (2c): Prepared from
2’,3’,5’-O-triacetyl-2-amino-6-chloropurine-9-riboside
treatment with 28% NH at room temperature followed by
flash chromatography (eluent: CH Cl /MeOH=11/2); yield:
93%. H NMR (400 MHz, DMSO-d ): d=3.55–3.65 (2 m,
and purified by flash chromatography, eluent:
1 13
Cl /MeOH=9/1; yield: 60%. H NMR, C NMR and
2
[41]
2
[38]
1
and
=13.8 min.
R
1
13
purified by preparative HPLC. H NMR and C NMR, see
Ref. ; ESI-MS: m/z=283 [M+H] ; HPLC: t =8.9 min.
[23]
+
[42]
by
R
[38]
1
-Methylguanosine (1e): Prepared according to Ref.
3
1
13
and purified by preparative HPLC. H NMR and C NMR,
see Ref. ; ESI-MS: m/z=298 [M+H] ; HPLC: t =
2
2
[37]
+
1
R
6
1
0.8 min.
2H, H-5’), 3.92 (m, 1H, H-4’), 4.13 (br s, 1H, H-3’), 4.48 (br
s, 1 H, H-2’), 5.04 (br s, 1H, HO-5’), 5.18 (br s, 1H, HO-3’),
5.48 (br s, 1H, HO-2’), 5.81 (d, J=5.7 Hz, H-1’), 6.97 (s, 2H,
[27]
8
-Bromoinosine (1f): See Ref. ; HPLC: t =10.6 min.
-Bromoguanosine (1g): See Ref. ; HPLC: t =13.2 min.
R
[39]
8
R
102
asc.wiley-vch.de
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 96 – 104

Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase
1
3
NH ), 8.38 (s, 1H, H-8); C NMR (100 MHz; DMSO-d ):
b) Preparative scale: To a suspension of 7-methylguano-
sine iodide (4) and the nucleobase in a molar ratio of 3 or 4
(see below and Scheme 2) in 50 mM phosphate buffer at
pH 7.5 (20 mL), 45.6 IU of Ah PNPII were added (80 mL,
570 IU/mL) and the resulting mixture was stirred at room
temperature. Aliquots (70 mL) were periodically withdrawn
from the reaction, diluted to 1 mL with a mixture of 50 mM
phosphate buffer pH 7.5 and glycerol (4:1), centrifuged
(10 kDa MWCO centrifugal filter devices, 5000 rpm, room
temperature, 4 min) and analyzed by HPLC. After 3 h the
mixture was heated at 608C for 30 min to deactivate the
enzyme and successively cooled to 408C until precipitation
of the 7-methylguanine (6) was complete. 7-Methylguanine
(6) was removed by filtration under vacuum and the solu-
tion containing the desired nucleoside was purified by prep-
arative HPLC and lyophilized.
2
6
d=61.70 (C-5’), 70.74 (C-3’), 74.06 (C-2’), 85.87 (C-4’), 87.25
C-1’), 124.01 (C-5), 141.66 (C-8), 150.00 (C-4), 154.55 (C-2),
60.29 (C-6); ESI-MS: m/z=324.2 [M+Na] ; HPLC: t =
(
1
+
R
1
4.3 min.
6
[43]
-Methoxypurine-9-riboside (2d): See Ref. ; HPLC: t =
R
1
3.5 min.
-Amino-6-methoxypurine-9-riboside (2e): See Ref.
HPLC: t =15.2 min.
-Thioinosine (3a): See Ref. ; HPLC (t )=13.8 min.
-Thioguanosine (3b): Prepared according to Ref.
C NMR (100 MHz; DMSO-d ): d=61.73 (C-5’), 70.75 (C-
’), 74.27 (C-2’), 85.76 (C-4’), 86.94 (C-1’), 128.82 (C-5),
48.34 (C-4), 153.64 (C-2), 175.59 (C-6); ESI-MS: m/z=
22.1 [M+Na] ; HPLC: t =11.0 min.
-Methylguanosine iodide (4): Prepared according to
H NMR (400 MHz, DMSO-d ): d=3.60–3.73 (2 m,
H, H-5’), 3.98 (m, 1H, H-4’), 4.01 (s, 3H, CH -N7), 4.16
m, 1H, H-3’), 4.37 (m, 1H, H-2’), 5.11 (t, J=5.2 Hz, 1H,
HO-5’), 5.32 (d, J=5.2 Hz, 1H, HO-3’), 5.63 (d, J=5.2 Hz,
H, HO-2’), 5.83 (d, J=3.6 Hz, 1H, H-1’), 7.21 (s, 2H,
[44]
2
;
R
[45]
6
6
R
[46]
13
6
3
1
3
+
R
7
Ref.
[32] 1
2-Amino-6-chloropurine-9-riboside (2c): Molar ratio sugar
donor/nucleobase=3/1. Using 180 mg of 7-methylguanosine
iodide (4, 0.42 mmol) and 25 mg of 2-amino-6-chloropurine
(0.14 mmol), 2c was obtained; yield: 39 mg (0.13 mmol, 93%
6
2
3
(
1
13
1
yield). H NMR, C NMR and ESI-MS, see above.
1
3
NH ), 9.36 (s, 1H, H-8), 11.68 (s, 1H, NH-1); C NMR
6-Methoxypurine-9-riboside (2d): Molar ratio sugar
donor/nucleobase=4/1.Using 240 mg of 7-methylguanosine
iodide (4, 0.56 mmol) and 21 mg of 6-methoxypurine
(0.14 mmol), 2d was obtained; yield: 28 mg (0.10 mmol,
2
(
(
100 MHz; DMSO-d ): d=36.33 (CH ), 60.88 (C-5’), 69.76
6
3
C-3’), 74.72 (C-2’), 86.19 (C-4’), 89.21 (C-1’), 108.12 (C-5),
36.78 (C-8), 149.68 (C-4), 153.83 (C-2), 156.11 (C-6); ESI-
MS: m/z=298 [M+H] ; HPLC: t =6.7 min.
1
+
1
13
71%). H NMR, C NMR and ESI-MS, see Ref.[43]
R
2
-Amino-6-(methylthio)purine-9-riboside (2g): Molar
ratio sugar donor/nucleobase=4/1. Using 240 mg of 7-meth-
ylguanosine iodide (4, 0.56 mmol) and 25 mg of 2-amino-6-
Phosphorolysis Experiments
(
4
methylthio)purine (0.14 mmol), 2g was obtained; yield:
1
Ah PNPII stability assay: In a final volume of 10 mL,
1 mg (0.13 mmol, 93%). H NMR (400 MHz, DMSO-d ):
6
50 mM phosphate buffer at pH 7.5 (8 mL) and glycerol
d=2.58 (s, 3H, -SCH ), 3.57–3.66 (2 m, 2H, H-5’), 3.83 (m,
(
2 mL) were mixed. After the addition of enzyme (100 mL),
3
1
H, H-4’), 4.12 (br s, 1H, H-3’), 4.47 (m, 1H, H-2’), 5.06 (t,
J=4.8 Hz, 1H, HO-5’), 5.14 (br s, 1H, HO-3’), 5.42 (br s,
H, HO-2’), 5.80 (d, J=6.0 Hz, 1H, H-1’), 6.53 (s, 2H,
the mixture was stirred at room temperature for 24 h. At
fixed times (1 min, 30 min, 1 h, 3.5 h, 6 h, 24 h), 1 mL of this
solution was submitted to activity assay (see above). The
enzyme completely maintained its activity after 24 h incuba-
tion.
1
1
3
NH ), 8.18 (s, 1H, H-8). C NMR (100 MHz; DMSO-d ):
d=11.25 (CH -S-C-6), 61.87 (C-5’), 70.84 (C-3’), 73.98 (C-
2
1
3
2
6
3
’), 85.79 (C-4’), 87.01 (C-1’), 124.85 (C-5), 139.21 (C-8),
General procedure of phosphorolysis: To a solution of
0 mM phosphate buffer at pH 7.5 and glycerol (4:1)
51.23 (C-4), 160.00 (C-2), 160.59 (C-6); ESI-MS: m/z=
5
+
36.2 [M+Na] .
(
20 mL) containing uracil (2.4 mg, 0.02 mmol) as internal
standard, the modified nucleoside (0.02 mmol) and 1.15 IU
of Ah PNPII were added. At fixed times (2, 4, 6, 8 and
30 min, 1 h, 3 h, 6 h), aliquots (0.2 mL) of this solution were
withdrawn, centrifuged (10 kDa MWCO centrifugal filter
devices, 5000 rpm at room temperature) for 4 min and ana- Acknowledgements
lyzed by HPLC (see Table 2).
This work was partially supported by Regione Lombardia –
Metadistretti Call 2007 (MD 2007 ID 4165). C.I.N.M.P.I.S.
Consorzio Interuniversitario Nazionale Metodologie e Proc-
essi Innovativi di Sintesi – Italy) is gratefully acknowledged
for the fellowship to C.D.S. We thank prof. A. M. Iribarren
Chemoenzymatic Synthesis of Purine-9-ribosides:
General Procedure
(
a) Analytical scale: To a solution of 50 mM phosphate
buffer at pH 7.5 and glycerol (4:1) (20 mL) containing 7-
methylguanosine iodide (4, 12 mg, 0.04 mmol) and the nu-
cleobase (0.01 mmol, see Scheme 2), 2.30 IU of Ah PNPII
were added. At fixed times (2, 4, 6, 8 and 30 min, 1 h, 3 h,
(
University of Quilmes, Buenos Aires, Argentina) for provid-
ing us with the Aeromonas hydrophila strain and G. Amati
for his help in the experimental.
6
h and 24 h), aliquots (0.2 mL) of this solution were with-
drawn, centrifuged (10 kDa MWCO centrifugal filter devi-
ces, 5000 rpm, room temperature) for 4 min and analyzed by References
HPLC (see Scheme 2). The produced nucleosides, i.e., 2c,
2
d, 2g and 2h were identified by comparison of their reten-
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