Site-Selective ribosylation of fluorescent nucleobase analogs using purine-Nucleoside phosphorylase as a catalyst: effects of point mutations

Article abstract of DOI:10.3390/molecules21010044

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- And N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- And N7-positions. The same mutant allows synthesis of the fluorescent N7-β-D-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- And N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-D-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

Full text of DOI:10.3390/molecules21010044

Article
Site-Selective Ribosylation of Fluorescent Nucleobase
Analogs Using Purine-Nucleoside Phosphorylase as a
Catalyst: Effects of Point Mutations
Alicja Stachelska-Wierzchowska 1,*, Jacek Wierzchowski 1, Agnieszka Bzowska 2 and
Beata Wielgus-Kutrowska 2
Received: 13 November 2015 ; Accepted: 9 December 2015 ; Published: 28 December 2015
Academic Editor: Mahesh K. Lakshman
1
Department of Physics and Biophysics, University of Varmia & Masuria in Olsztyn, 4 Oczapowskiego St.,
10-719 Olsztyn, Poland; jacek.wie@uwm.edu.pl
Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93,
2
02-089 Warsaw, Poland; abzowska@biogeo.uw.edu.pl (A.B.); beata@biogeo.uw.edu.pl (B.W.-K.)
*
Correspondence: alicja.stachelska@uwm.edu.pl; Tel.: +48-89-523-3406; Fax: +48-89-523-3861
Abstract: Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and
2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9,
N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations
in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP
gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and
N7-positions. The same mutant allows synthesis of the fluorescent N7-β-D-ribosyl-8-azaguanine.
The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of
8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines
can be analytically useful, as illustrated by N7-β-D-ribosyl-2,6-diamino-8-azapurine, which is a
good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the
N8-riboside is selective to the E. coli enzyme.
Keywords: fluorescent nucleosides; enzymatic ribosylation; 8-azapurines; purine nucleoside
phosphorylase
1. Introduction
Purine-nucleoside phosphorylase (PNP, E.C.2.4.2.1) is a key enzyme of purine metabolism,
important, inter alia, for the proper activity of the immune system in mammals [
inhibitors of PNP, like immucillin H (forodesine) and some analogs [ ], are clinically approved
to treat lymphomas [ ], and others are considered as potential anti-parasitic and anti-malarial
drugs [ ]. Bacterial forms of PNP are of interest because they can be used as a suicidal-gene in cancer
chemotherapy [ ]. Besides this, PNP is used as a catalyst in the gram-scale preparative ribosylation of
purines and purine analogs [ 11], thanks to the reverse (synthetic) pathway of the phosphorolytic
1–3]. Potent
4
4–6
4,7
8
9–
process. Nucleoside analogues are widely applied as pharmaceuticals and as biochemical probes for
enzymological studies [5–7,9–13].
In the preceding papers [14,15], we have demonstrated that enzymatic ribosylation of some
8-azapurines leads not only to the canonical nucleoside analogs, but also to non-typical, and highly
fluorescent ribosides, ribosylated at the N7- and N8-positions (see Figure 1 below). Now we present
a kinetic analysis of these processes, with application of several wild and mutated forms of PNP as
catalysts. We will demonstrate a considerable selectivity of the 8-azapurine ribosylation sites with
various PNP forms, and a remarkable sensitivity of the ribosylation process to point mutations at the
Molecules 2016, 21, 44; doi:10.3390/molecules21010044
www.mdpi.com/journal/molecules

Molecules 2016, 21, 44
2 of 10
critical active site residue. Finally, we present an example of potential analytical application of the
obtained compounds to blood PNP determination.
Figure 1. Ribosylation of 8-azaguanine with
α-D-ribose-1-phosphate: possible reaction products.
The purine numbering is maintained for simplicity. Only the major tautomeric form of 8-azaguanine
(N(9)H) is shown.
2. Results and Discussion
In comparison with natural purines, their 8-aza analogues are not as good PNP substrates, but
their ribosides are highly fluorescent and therefore can be utilized as probes in enzymology or clinical
investigations [11]. Our aim was to identify those forms of PNP which can be used as catalyst in the
effective and selective enzymatic syntheses of these ribosides.
We have investigated bacterial (E. coli) and mammalian (calf) forms of PNP, as the most widely
accessible, and representing two main classes of the enzyme, as well as their mutated forms, which
has been previously shown to express altered activity of the phosphorolytic process [1]. In particular,
the N243D mutant of the calf and human enzymes is known to catalyze the phosphorolysis not only of
Guo and Ino (as do the wild forms), but also of Ado [
the E. coli PNP, D204N, obtained recently [17], will be also interesting as a potential catalyst.
In the ribosylation reactions, we used -D-ribose-1-phosphate, prepared enzymatically
1,16]. We expected that the analogous mutant of
α
(see Section 3 for details), as a ribosyl donor [14]. Additionally, we have also measured kinetics
of phosphorolysis of 8-azapurine ribosides in the phosphate buffer.
2.1. Ribosylation of 8-Azaguanine and 2,6-Diamino-8-azapurine
Ribosylation of 8-azaguanine (8-azaGua) and 2,6-diamino-8-azapurine (8-azaDaPur) was followed
spectrophotometrically, and the reaction products analyzed using HPLC separation coupled with
spectrophotometric and fluorimetric detectors. The products were identified by comparing their
spectral properties with those published earlier [18–20]. The non-typical ribosides of both substrates are
spectrally distinct from the N9-nucleosides in that the UV spectra of the formers are quite red-shifted,
allowing relatively easy detection at 315 nm. Typical elution profile from the C8-column is shown on
Figure 2.

Molecules 2016, 21, 44
3 of 10
Figure 2. The HPLC elution profile for the mixture obtained from the ribosylation of 8-azaguanine
with -D-ribose-1-phosphate as a ribose donor, catalyzed by the N243D mutant of the calf PNP. The
Kromasil C8 column (5 250 10 mm) was used, and the reaction mixture eluted with water
α
µm
ˆ
ˆ
(10 min) followed by 0%–30% methanol gradient. The first peak (~6 min retention time) was identified
as unreacted 8-azaguanine.
As pointed earlier, enzymatic ribosylation of 8-azaguanine goes fairly rapidly, when catalyzed by
calf PNP [11], and the only product of this process is N9-riboside. By contrast, the mutated (N243D)
form of the enzyme gives as a major product N7-riboside, although the overall rate of this process
is quite slow. Similar qualitative differences can be observed for 8-azaDaPur ribosylation: while the
wild-type enzyme gives a mixture of N8 and N7 forms, the application of the N243D mutant gives
mainly N7, some N9- and virtually no N8-riboside (see Table 1).
Table 1. Kinetic parameters for enzymatic ribosylation of selected 8-azapurines in 25 mM HEPES buffer,
pH 6.6, by α-D-ribose-1-phosphate, using various forms of PNP (nd = not determined). Standard errors
are estimated to be ~15%.
Approximate Product
Ratio: N9-riboside:
N8-riboside:N7-riboside
Vmax
(Relative) *
Substrate/Enzyme
Km (µM)
8-azaGua/calf PNP-wt
8-azaGua/calf PNP-N243D
8-azaGua/E. coli PNP-wt
8-azaGua/E. coli PNP-D204N
8-azaDaPur/calf PNP-wt
~90
>100
>200
nd
21
>0.2 ***
~1 ***
traces
~1
1:0:0
1:0:2
20:1:0
predominantly N9
0:1:1 **
60
8-azaDaPur/calf PNP-N243D
8-azaDaPur/E. coli PNP-wt
8-azaDaPur/E. coli PNP-D204N
35
>200
>200
0.6
~3 ***
~0.4 ***
1:0:3
1:2:0
10:1:0
* relative to guanine ribosylation under the same conditions (=100); ** this ratio is dependent on the reaction
progress; *** reaction rates measured at 200 µM substrate.
The E. coli PNP (wild type) gives predominantly N9-riboside of 8-azaguanine, with some
participation of a highly fluorescent N8-form. Similar specificity was obtained using the D204N
mutant, although the reaction was much slower. With 8-azaDaPur as a substrate, the N8-riboside
becomes a dominant ribosylation product for the wild-type enzyme, while the mutant gave mainly
N9-nucleoside. Ribosylation rates are typically lower for the mutated enzymes, comparing with the

Molecules 2016, 21, 44
4 of 10
wild types. The Km values, determined by standard methods, are lower for the calf enzymes (Table 1),
while the E. coli PNP was not saturated under the applied conditions.
The E. coli PNP is known to ribosylate adenine and 8-azaadenine [1,11]. In both cases, the
N9-ribosides were the only detectable ribosylation products. We have found that the D204N mutant
reacting with the latter substrate does produce also a non-typical product with red-shifted UV spectra,
tentatively identified as N8-riboside (data not shown).
The altered specificity of the PNP mutants towards some of the purine analogs has been observed
in other laboratories [21]. It can be related to differences in binding modes of 8-azapurine bases in the
PNP active site, including binding in the “upside-down” position (rotation by 180 deg when compared
with standard binding mode). In fact, such an “upside-down” binding mode was already observed
in the crystal structure of calf PNP complexed with N7-acycloguanosine inhibitor [22]. The altered
binding geometry can possibly be associated also with prototropic tautomerism or proton affinity
alterations [23–26]. This illustrates remarkable plasticity of the mammalian and bacterial PNP active
site, which may be profited from in designing new more potent and better membrane-permeable PNP
inhibitors—potential pharmaceutical agents [1–7].
There is a considerable progress in enzymatic synthesis of nucleoside analogues on a gram
scale using PNP as a catalyst [
is a possibility to alter ribosylation specificity of PNP by point mutations, an effect noted also by
other authors [21]. Although the non-typical ribosides are less biologically active than their N9-
9,10,27–30]. This work demonstrates that in some substrates there
β
isomers, they sometimes exhibit interesting spectral properties and could be applied as fluorescent
probes [14,15,21].
2.2. Phosphorolysis of 8-Azaguanine and 2,6-Diamino-8-azapurine Ribosides
On the basis of the micro-reversibility principle, it can be expected that mutations in the active
site of PNP can alter the phosphorolytic processes as well. We have therefore purified the 8-azapurine
ribosides and examined them as potential PNP substrates in the phosphate buffer, with results
summarized in Table 2. The phosphorolytic reactions were followed spectrophotometrically and, if
possible, also fluorimetrically, and the results are summarized in Table 2.
Table 2. Kinetic parameters for enzymatic phosphorolysis of selected ribosides in 25 mM phosphate
buffer, pH 6.5, at 25 ˝C, catalyzed by various forms of PNP. Errors about 15%.
Substrate
Enzyme (wt = Wild Type)
Km (µM)
Vmax (Relative) *
N7-β-D-ribosyl-Gua **
N7-β-D-ribosyl-Gua **
N7-ribosyl-8-azaGua
N7-ribosyl-8-azaGua
N7-ribosyl-8-azaDaPur
N7-ribosyl-8-azaDaPur
N7-ribosyl-8-azaDaPur
N7-ribosyl-8-azaDaPur
N8-ribosyl-8-azaDaPur
N8-ribosyl-8-azaDaPur
N9-ribosyl-8-azaDaPur
calf PNP-wt
E. coli-wt
calf PNP-wt
calf PNP-N243D
calf PNP-wt
calf PNP-N243D
E. coli PNP-wt
E. coli PNP-D204N
E. coli PNP-wt
E. coli PNP-D204N
E. coli PNP-wt
27
~450
nd
nd
52
>50
~80
nd
7
nd
0.6
33
>0.13
>1.5
~20
>60
~1.7
~0.7 ***
1.1
~1.6 ***
~0.02
~20
* relative to guanosine phosphorolysis under the same conditions (=100); ** data from ref. [1]; *** rate with
40 µM substrate.
8-Azaguanosine was reported to be a very weak substrate for mammalian PNP [11]. Similarly,
only traces of activity of the calf PNP towards N7-riboside were detected. One possible reason can be
unfavorable equilibrium of the phosphorolytic process, which for N9-riboside was estimated to be
~300 in favor of nucleoside synthesis, compared to ~50 for natural purines [11]. The mutated form of
the calf enzyme is somewhat more active (Table 2).
As mentioned earlier, there is a considerable specificity in the phosphorolytic pathway in both
calf and E. coli PNP in relation to 8-azaDaPur ribosides [15]. There was no detectable activity towards

Molecules 2016, 21, 44
5 of 10
N9-riboside with the calf PNP, and only residual with the E. coli enzyme. By contrast, the N8-riboside
is quite effectively phosphorolysed by the wild type E. coli PNP, but not by the mutant (see Table 2).
Worth noting is low Km value for this process (Table 2), contrasting with very high Km observed in the
synthetic reaction (see Table 1).
The N7-riboside of 8-azaDaPur seems to be quite rapidly and specifically phosphorolysed by
calf PNP [15]. The mutation at Asn243 markedly accelerates this process (see Table 2), while virtually
no activity towards N8- or N9-ribosides was detected using both wild and mutated types of PNP.
This finding is somewhat surprising in view of the fact that N8-ribosides are produced in the reverse
(synthetic) reaction catalyzed by wild (but not the mutated) form of calf PNP (see previous sections).
Although the spectrum of non-typical substrates of mammalian, and especially bacterial forms of
PNP is broad [
1
,
9
,
11], recent years brought much more examples of applications of these enzymes to
27 30]. But the exact
synthesis and phosphorolysis of biologically interesting nucleoside analogs [10
,
–
prediction of the substrate preferences of various forms is at present difficult [31], we think that large
variability of natural PNP and possibility of mutations in the active site can offer new potentially
useful catalyst for synthetic procedures in nucleoside chemistry.
2.3. Fluorescence of 8-Azaguanine and 2,6-Diamino-8-azapurine Ribosides and Potential Applications
Fluorescent nucleoside analogues are widely studied because of potential applications in
enzymology [11
measurable fluorescence in aqueous medium at room temperature, which was in several instances
applied to mechanistic and analytical studies [11 34 35]. The highest yields of fluorescence are observed
in 8-azaxanthine and 2,6-diamino-8-azapurine, as well as in some N-alkyl derivatives of these [11 36].
,32,33]. It has been reported that many 8-azapurines and their nucleosides exhibit
,
,
,
Nucleosides of adenine and hypoxanthine are more fluorescent than the parent bases [13], and
N8-ribosides of many 8-azapurines, including N8-ribosyl-8-azaguanine and N8-ribosyl-8-azaDaPur,
exhibit yields comparable to the best fluorophores known [11].
2.3.1. Fluorescence of 2,6-Diamino-8-azapurine Ribosides
Ribosylation of 2,6-diamino-8-azapurine leads to at least three fluorescent ribosides (Table 3). The
N9-riboside is very highly fluorescent, but its fluorescence is generally similar, in terms of λmax and
decay time, to that of the free base [11]. By contrast, fluorescence of N7- and N8-ribosides is red-shifted
by ~70 nm and thanks to this shift their cleavage via phosphorolysis (or hydrolysis) leads to a high
fluorogenic effect at ~360 nm, which can be analytically useful (see next section).
Table 3. Ionization constants (pKa values) and spectral parameters for neutral and ionic forms of
the selected 8-azapurine ribosides and free bases. The UV spectral data are compiled from the
literature [11,18,20] and fluorescence parameters determined in this and previous [11,15] works.
Form *
(pH)
UV Absorption
Fluorescence
Compound
pKa
ε
(M´1¨ cm´1
)
λ
(nm)
λ
(nm)
max
φ
τ (ns)
max
max
9-β-D-ribofuranosyl-8-azaDaPur 2.9
8-β-D-ribofuranosyl-8-azaDaPur 4.9
7-β-D-ribofuranosyl-8-azaDaPur 3.95
n (7)
c (2)
n (7)
c (2.7)
n (7)
c (2)
285
10,800
8100
8200
13,200
~5500
~12,000
368
0.9
nd **
0.41
nd **
0.063
nd **
6
283
313
264
314
258
360
430
430
420
420
nd **
10.6
nd **
1.5; 0.45
nd **
3.7;
8-azaDaPur (free base)
7.7
n (6)
280
8500
365
0.40
7.5; 0.2
9-β-D-ribofuranosyl-8-azaGua
7-β-D-ribofuranosyl-8-azaGua
8-azaGua (free base)
8.05
n (5)
ma (10)
n (5)
ma (10)
n (4.5)
256
278
302
304
249
12,900
11,700
4900
5100
11,200
347
362
410
420
395
~0.01
0.55
~0.04
~0.03
0.05–0.33
~0.1
5.6
nd **
nd **
6.2
7.4
6.5
* n—neutral form; c—cation; ma—monoanion; ** nd—not determined.

Molecules 2016, 21, 44
6 of 10
It is of interest that protonation of N7- and N8-ribosides, which leads to a significant blue shift in
the UV spectra (Table 3), apparently does not alter the observed fluorescence band. This is undoubtedly
due to a large shift in the acid-base equilibrium in the excided state (pK*), as compared to ground-state
(pKa), so upon excitation the protonated 8-azapurine moiety undergoes rapid deprotonation. This
process is observed also in acidified alcohols, where dual fluorescence can be observed, especially for
the N8-riboside (data not shown), an analogous effect reported earlier for the N8-methyl derivative [36].
It must be stressed that the strong fluorescence of 8-azaDaPur and its ribosides is sensitive to buffer
concentration, isotope exchange and other environmental factors [36], partially related to excited-state
proton transfer reactions and relatively long fluorescence decay times (Table 3). This creates some
difficulty in analytical applications, which can be overcome by using internal concentration standards
(e.g., purified products of enzymatic reactions at standardized concentrations).
2.3.2. Blood PNP Determination Using Fluorogenic Ribosides
It has been reported earlier that 7-
β-D-ribofuranosyl-8-azaDaPur is a highly fluorogenic substrate
for calf PNP in phosphate buffer [15]. Human PNP is similar to the calf enzyme [
1], and we have
therefore verified a possibility to detect PNP activity in human blood using the same substrate.
The experiment was run with 1000-fold diluted, lysed human blood in 25 mM phosphate, and in
a phosphate-free 20 mM HEPS buffer, pH 6.6, as a control. Ca. 16 µM N7-riboside was used as
a substrate. Virtually no fluorescence change was observed in the phosphate-free buffer (data not
shown), but in the presence of phosphate the appearance of an emission band at 365 nm indicated free
base production (Figure 3, Table 3).
Figure 3. Fluorescence changes during the incubation of 16 µM N7-ribosyl-8-azaDaPur in 1000-diluted
blood in the presence of 25 mM phosphate. Spectra, excited at 300 nm, were recorded every 5 min, and
after 60 min an aliquot of the purified calf PNP was added, and fluorescence recorded after next 3 min
(dotted line, 5-fold diminished relative to remaining curves). Note that the first (lowest) curve reflects
blood fluorescence background, and minimum at 415 nm is due to the re-absorption by hemoglobin.
The apparent Km for this process was rather high, ~90
fluorogenic effect (Figure 3) can undoubtedly be enhanced by optimizing assay conditions. Although
phosphorolysis of 7- -D-ribofuranosyl-8-azaDaPur is slow in comparison to the best PNP substrates,
µM (data not shown), and the presented
β
like guanosine or 7-methylguanosine [11], the fluorogenic effect is large thanks to high fluorescence
yield of the product (~0.4), and the present assay, in its optimized version, may be more sensitive and
simpler than other assays proposed for this enzyme [1,37].

Molecules 2016, 21, 44
7 of 10
Of special interest is high selectivity of the fluorogenic ribosides of 8-azaDaPur to various forms
of PNP, namely, mammalian (N7-ribosides) and bacterial (N8-ribosides), particularly in view of recent
applications of bacterial (E. coli) PNP as a suicidal-gene in cancer chemotherapy [8]. These two forms
of PNP can likely be assayed selectively in the same blood sample, with no pre-purification necessary.
3. Experimental Section
Recombinant E. coli and calf spleen PNP, as well as mutated form of these, were obtained and
purified as described elsewhere [17
,
38]. Stock solutions (0.1–1.7 mM per subunit) were stored frozen at
˝
´20 C and diluted prior to experiments. 2,6-Diamino-8-azapurine sulfate, N7-methylguanosine
(m7Guo) and 8-azaguanine were from Sigma-Aldrich (St. Louis, MO, USA), the latter was
re-crystallized as a monosodium salt. N7-methylguanosine was used without further purification.
Fluorescence was measured on a Varian Eclipse instrument (Varian Corp., Palo Alto, CA, USA),
and UV absorption kinetic experiments on a Cary 300 (Varian). All buffers were of analytical grade
and displayed no fluorescence background.
α
-D-Ribose-1-phosphate (100 mM solution in 100 mM HEPES buffer, pH ~7.2) was prepared
enzymatically from the N7-methylguanosine and inorganic phosphate, using the modified procedure
of Krenitsky et al. [39]. The recombinant calf PNP (~2 g per 1 mL reaction volume) was used as catalyst,
µ
and the reaction progress was monitored fluorimetrically [40]. Alternatively, the N243D mutant can
be used as a more selective towards m7Guo [41]. The second reaction product, N7-methylguanine,
was removed in nearly 97% by spontaneous crystallization and filtration. The phosphorylated ribose
˝
solution was stored at
´
20 C and assayed using previously described fluorimetric method [37]. It was
found that 1-year storage caused hydrolysis of not more than 20% of the compound.
Synthetic reactions were carried out on a milligram scale as previously described [14], typically in
1 mL volume, in HEPES buffer. Enzyme concentrations were 1–10
~5 mM. After 24 h reaction mixtures were frozen.
µg/mL, and ribose-1-phosphate
Reaction products were analyzed by the analytical reverse-phase HPLC on a UFLC system from
Shimadzu (Kyoto, Japan) equipped with UV (diode-array) detection at 280 nm and 315 nm. The
column used was a Kromasil reversed-phase analytical C8 column (250
ˆ
4.6 mm, 5-µm particle size).
For product separation, an analogous semi-preparative column was used. The eluent was deionized
water (10 min), followed by a linear gradient from 0% to 30% methanol (60 min). The reactions were
carried out at pH ~6.5, and samples containing 8-azaguanine ribosides were acidified to ~5 prior to the
HPLC analysis.
Kinetic parameters of the enzymatic reactions were calculated using linear regression analysis of
the double-reciprocal plots. Substrate concentration in the kinetic experiments ranged typically from
1 to ~200 µM, and enzyme concentrations were adjusted so that the reaction rates were in the range
0.1 to ~5 uM/min. Fluorescence measurements were conducted in semi-micro cuvettes (pathlength
0.4 cm, volumen 1 mL) to diminish the inner filter effect.
Acknowledgments: This work has been supported by the Ministry of Higher Education of Poland, grant #N
N301 044939 and by the University of Varmia & Masuria in Olsztyn, internal grant #17.610.006-110. The authors
are indebted to Goran Mikleusevic for a sample of the D204N mutated form of the E. coli PNP and to Mariusz
Szabelski for determination of fluorescence lifetimes.
Author Contributions: A.S.-W. and J.W. are responsible for kinetic and spectroscopic measurements, and A.S.-W.
additionally for HPLC analysis. Enzyme cloning and purifications has been done by A.M.B. and B.W.K. This
paper was written jointly by all the authors.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
PNP, purine-nucleoside phoshorylase; 8-azaGua, 8-azaguanine; 8-azaDaPur, 2,6-diamino-8-azapurine.

Molecules 2016, 21, 44
8 of 10
References
1.
Bzowska, A.; Kulikowska, E.; Shugar, D. Purine nucleoside phosphorylases: Properties, functions, and
clinical aspects. Pharmacol. Ther. 2000, 88, 349–425. [CrossRef]
2.
Grunebaum, E.; Cohen, A.; Roifman, C.M. Recent advances in understanding and managing adenosine
deaminase and purine nucleoside phosphorylase deficiencies. Curr. Opin. Allergy Clin. Immunol. 2013, 13,
630–638. [CrossRef] [PubMed]
3.
4.
5.
6.
7.
Edwards, P.N. A kinetic, modeling and mechanistic re-analysis of thymidine phosphorylase and some
related enzymes. J. Enzym. Inhib. Med. Chem. 2006, 21, 483–499. [CrossRef] [PubMed]
Taylor Ringia, E.A.; Schramm, V.L. Transition states and inhibitors of the purine nucleoside phosphorylase
family. Curr. Top. Med. Chem. 2005, 5, 1237–1258. [CrossRef] [PubMed]
Robak, P.; Robak, T. Older and new purine nucleoside analogs for patients with acute leukemias.
Cancer Treat. Rev. 2013, 39, 851–861. [CrossRef] [PubMed]
Al-Kali, A.; Gandhi, V.; Ayoubi, M.; Keating, M.; Ravandi, F. Forodesine: Review of preclinical and clinical
data. Future Oncol. 2010, 6, 1211–1217. [CrossRef] [PubMed]
De Jersey, J.; Holý, A.; Hocková, D.; Naesens, L.; Keough, D.T.; Guddat, L.W. 6-oxopurine
phosphoribosyltransferase: A target for the development of antimalarial drugs. Curr. Top. Med. Chem. 2011
11, 2085–2102. [CrossRef] [PubMed]
,
8.
9.
Portsmouth, D.; Hlavaty, J.; Renner, M. Suicide genes for cancer therapy. Mol. Asp. Med. 2007, 28, 4–41.
[CrossRef] [PubMed]
Mikhailopulo, I.A.; Miroshnikov, A.I. Biologically important nucleosides: Modern trends in biotechnology
and application. Mendeleev Commun. 2011, 21, 57–68. [CrossRef]
10. Zhou, X.; Mikhailopulo, I.A.; Cruz Bournazou, M.N.; Neubauer, P. Immobilization of thermostable
r
nucleoside phosphorylases on MagReSyn epoxide microspheres and their application for the synthesis of
2,6-dihalogenated purine nucleosides. J. Mol. Catal. B Enzym. 2015, 115, 119–127. [CrossRef]
11. Wierzchowski, J.; Antosiewicz, J.M.; Shugar, D. 8-Azapurines as isosteric purine fluorescent probes for
nucleic acid and enzymatic research. Mol. BioSyst. 2014, 10, 2756–2774. [CrossRef] [PubMed]
12. Giorgi, I.; Scartoni, V. 8-Azapurine nucleus: A versatile scaffold for different targets. Mini Rev. Med. Chem.
2009, 9, 1367–1378. [CrossRef] [PubMed]
13. Wierzchowski, J.; Wielgus-Kutrowska, B.; Shugar, D. Fluorescence emission properties of 8-azapurines
and their nucleosides, and application to the kinetics of the reverse synthetic reaction of PNP.
Biochim. Biophys. Acta 1996, 1290, 9–17. [CrossRef]
14. Stachelska-Wierzchowska, A.; Wierzchowski, J.; Wielgus-Kutrowska, B.; Mikleuševic´, G. Enzymatic synthesis
of highly fluorescent 8-azapurine ribosides using purine-nucleoside phosphorylase reverse reaction: Variable
ribosylation sites. Molecules 2013, 18, 12587–12598. [CrossRef] [PubMed]
15. Wierzchowski, J.; Stachelska-Wierzchowska, A.; Wielgus-Kutrowska, B.; Mikleuševic´, G. Two fluorogenic
substrates for purine-nucleoside phosphorylase, selective for mammalian and bacterial forms of the enzyme.
Anal. Biochem. 2014, 446, 25–27. [CrossRef] [PubMed]
16. Stoeckler, J.D.; Poirot, A.F.; Smith, R.M.; Parks, R.E., Jr.; Ealick, S.E.; Takabayashi, K.; Erion, M.D. Purine
nucleoside phosphorylase. 3. Reversal of purine base specificity by site-directed mutagenesis. Biochemistry
1997, 36, 11749–11756. [CrossRef] [PubMed]
17. Mikleuševic´, G.; Štefanic´, Z.; Narczyk, M.; Wielgus-Kutrowska, B.; Bzowska, A.; Luic´, M. Validation of the
catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies.
Biochimie 2011, 93, 1610–1622. [CrossRef] [PubMed]
18. Montgomery, J.A.; Shortnacy, A.T.; Secrist, J.A., III. Synthesis and biological evaluation of
2-fluoro-8-azaadenosine and related compounds. J. Med. Chem. 1983, 26, 1483–1489. [CrossRef] [PubMed]
19. Seela, F.;
Lampe, S. 8-Aza-21-deoxyguanosine and related 1,2,3-triazolo[4,5-d]pyrimidine
21-deoxyribofuranosides. Helv. Chim. Acta 1993, 72, 2388–2397. [CrossRef]
20. Elliott, R.D.; Montgomery, J.A. Analogues of 8-azaguanosine. J. Med. Chem. 1976, 19, 1186–1191. [CrossRef]
21. Ye, W.; Paul, D.; Gao, L.; Seckute, J.; Sangaiah, R.; Jayaraj, K.; Zhang, Z.; Kaminski, P.A.; Ealick, S.E.;
Gold, A.; et al
.
Ethenoguanines undergo glycosylation by nucleoside 21-deoxyribosyltransferases at
non-natural sites. PLoS ONE 2014, 9, e115082. [CrossRef] [PubMed]

Molecules 2016, 21, 44
9 of 10
22. Luic´, M.; Koellner, G.; Shugar, D.; Saenger, W.; Bzowska, A. Calf spleen purine nucleoside phosphorylase:
Structure of its ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate anion.
Acta Crystallogr. D57 2001, 57, 30–36. [CrossRef]
23. Kierdaszuk, B.; Modrak-Wójcik, A.; Wierzchowski, J.; Shugar, D. Formycin A and its N-methyl analogues,
specific inhibitors of E. coli purine nucleoside phosphorylase: Induced tautomeric shift on binding to enzyme,
and enzyme
[CrossRef]
Ñ
ligand fluorescence resonance energy transfer. Biochim. Biophys. Acta 2000, 1476, 109–128.
24. Pyrka, M.; Maciejczyk, M. Theoretical study of tautomeric equillibria of 2,6–diamino-8-azapurine and
8-aza-isoguanine. Chem. Phys. Lett. 2015, 627, 30–35. [CrossRef]
25. Wierzchowski, J.; Bzowska, A.; Ste˛pniak, K.; Shugar, D. Interactions of calf spleen purine nucleoside
phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: Implications for the reaction
mechanism. Z. Naturforsch. 2004, 59, 713–725. [CrossRef]
26. Gasik, Z.; Shugar, D.; Antosiewicz, J.M. Resolving differences in substrate specificities between human
and parasite phosphoribosyltransferases via analysis of functional groups of substrates and receptors.
Curr. Pharm. Des. 2013, 19, 4226–4240. [CrossRef] [PubMed]
27. Zhou, X.; Szeker, K.; Jiao, L.Y.; Oestreich, M.; Mikhailopulo, I.A.; Neubauer, P. Synthesis of 2,6-dihalogenated
purine nucleosides by thermostable nucleoside phosphorylases. Adv. Synth. Catal. 2015, 357, 1237–1244.
[CrossRef]
28. Fateev, I.V.; Kharitonova, M.I.; Antonov, K.V.; Konstantinova, I.D.; Stepanenko, V.N.; Esipov, R.S.; Seela, F.;
Temburnikar, K.W.; Seley-Radtke, K.L.; Stepchenko, V.A.; et al. Recognition of Artificial Nucleobases by E. coli
Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides.
Chem. Eur. J. 2015, 21, 13401–13419. [CrossRef] [PubMed]
29. Calleri, E.; Cattaneo, G.; Rabuffetti, M.; Serra, I.; Bavaro, T.; Massolini, G.; Speranza, G.; Ubiali, D.
Flow-Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from
Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification. Adv. Synth. Catal.
2015, 357, 2520–2528. [CrossRef]
30. Calleri, E. Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme
reactor for biocatalytic applications. J. Chromatogr. B 2014, 968, 79–86. [CrossRef] [PubMed]
31. Štefanic´, Z.; Mikleuševic´, G.; Narczyk, M.; Wielgus-Kutrowska, B.; Bzowska, A.; Luic´, M. Still a long
way to fully understanding the molecular mechanism of Escherichia coli Purine Nucleoside Phosphorylase.
Croat. Chem. Acta 2013, 86, 117–127. [CrossRef]
32. Sinkeldam, R.W.; Greco, N.J.; Tor, Y. Fluorescent analogs of biomolecular building blocks: Design, properties,
and applications. Chem. Rev. 2010, 110, 2579–2619. [CrossRef] [PubMed]
33. Pollum, M.; Martínez-Fernández, L.; Crespo-Hernández, C.E. Photochemistry of nucleic acid bases and their
thio- and aza-analogues in solution. Top. Curr. Chem. 2015, 355, 245–355. [PubMed]
34. Liu, L.; Cottrell, J.W.; Scott, L.G.; Fedor, M.J. Direct measurement of the ionization state of an essential
guanine in the hairpin ribozyme. Nat. Chem. Biol. 2009, 5, 351–357. [CrossRef] [PubMed]
35. Cottrell, J.W.; Scott, L.G.; Fedor, M.J. The pH dependence of hairpin ribozyme catalysis reflects ionization of
an active site adenine. J. Biol. Chem. 2011, 286, 17658–17664. [CrossRef] [PubMed]
36. Wierzchowski, J.; Me˛dza, G.; Szabelski, M.; Stachelska-Wierzchowska, A. Properties of 2,6-diamino-8-
azapurine, a highly fluorescent purine analog and its N-alkyl derivatives: Tautomerism and excited-state
proton transfer reactions. J. Photochem. Photobiol. A 2013, 265, 49–57. [CrossRef]
37. Wierzchowski, J.; Ogiela, M.; Iwan´ska, B.; Shugar, D. Selective fluorescent and fluorogenic substrates for
purine-nucleoside phosphorylases from various sources, and direct fluorimetric determination of enzyme
levels in human and animal blood. Anal. Chim. Acta 2002, 472, 63–74. [CrossRef]
38. Breer, K.; Girstun, A.; Wielgus-Kutrowska, B.; Staron, K.; Bzowska, A. Overexpression, purification and
characterization of functional calf purine nucleoside phosphorylase (PNP). Protein Expr. Purif. 2008, 61,
122–130. [CrossRef] [PubMed]
39. Krenitsky, T.A.; Koszalka, G.W.; Tuttle, J.V. Purine nucleoside synthesis, an efficient method employing
nucleoside phosphorylases. Biochemistry 1981, 20, 3615–3621. [CrossRef] [PubMed]
40. Kulikowska, E.; Bzowska, A.; Wierzchowski, J.; Shugar, D. Properties of two unusual, and
fluorescent substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine.
Biochim. Biophys. Acta 1986, 874, 355–366. [CrossRef]

Molecules 2016, 21, 44
10 of 10
41. Wielgus-Kutrowska, B. Division of Biophysics, Institute of Experimental Physics; University of Warsaw: Warsaw,
Poland, Unpublished Work.
Sample Availability: Samples of the compounds are available from the authors.
©
2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).