Kinetic parameters and recognition of thymidine analogues with varying functional groups by thymidine phosphorylase

Article abstract of DOI:10.1016/j.bmc.2008.01.038

Thymidine phosphorylase (TP, EC 2.4.2.4) recognized the structure of the substrate with high specificity, via both the base and the ribosyl moieties. The replacement of 3′-OH of thymidine markedly influenced its catalytic activity with TP. The conversion

Full text of DOI:10.1016/j.bmc.2008.01.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3866–3870
Kinetic parameters and recognition of thymidine analogues
with varying functional groups by thymidine phosphorylase
Akihiko Hatano,* Aiko Harano, Yoshikatsu Takigawa, Yasuhiro Naramoto,
Keisuke Toda, Yuuichi Nakagomi and Hideyuki Yamada
Department of Materials and Life Science, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi,
Shizuoka 437-8555, Japan
Received 27 November 2007; revised 21 January 2008; accepted 23 January 2008
Available online 12 February 2008
Abstract—Thymidine phosphorylase (TP, EC 2.4.2.4) recognized the structure of the substrate with high specificity, via both the
base and the ribosyl moieties. The replacement of 30-OH of thymidine markedly influenced its catalytic activity with TP. The con-
version of pyrimidine nucleosides with modified base moieties to the corresponding 1-phosphate form was poor. The leaving group
activity decreased with an increase in aromaticity of the pyrimidine base moiety, because of increased difficulty in polarizing the base
by the amino acids local to the active site. The replacement of 30 and 50 functional groups tended to decrease the reaction rate and
the percentage conversion with TP. In particular the ribosyl 30 hydroxyl group was structurally important for the binding of the
substrate by the enzyme. The kinetic assay clearly showed high Km and low Vmax values on replacing the 30 hydroxyl group with
hydrogen.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
(2-iminopyrrolidinyl)methyl]uracil) HCl in an active
conformation.15 This inhibitor has been proposed as a
transition-state analogue of TP, assuming that the tran-
sition-state structure is oxonium-like and that the pro-
tonated pyrrolidine is an oxonium mimic. However,
this structure of TP complexed with an inhibitor offers
no direct insights as to the basis of specific interactions
between TP and the ribosyl moiety.
Thymidine phosphorylase (TP, EC 2.4.2.4) is a unique
metabolic control enzyme, which converts thymidine
to 2-deoxy-D-ribose-1a-phosphate and thymine base in
phosphate buffer (Scheme 1). TP can effect the transgly-
cosylation of pyrimidine nucleosides, and thus it is
available to synthesize unnatural nucleosides.1,2 Addi-
tionally, it is known that TP is associated with tumor-
dependent angiogenesis.3–5 Inhibition of this enzyme
could potentially be useful in treating tumors depending
on the nucleoside salvage pathway for their proliferation
Here, we report the reactivity and kinetics of thymidine
phosphorylase (from E. coli) using thymidine analogues.
Our research proves the influence of the functional
groups and the ribosyl structure on the ability of TP
to discriminate between substrates, and it will provide
the basis for further drug design studies with the aim
of increasing inhibitor potency and specificity.
and vascularization6–10
.
Thymidine phosphorylase is a dimeric enzyme made up
of two identical subunits.11 Human TP shares 39% of its
sequence identity with Escherichia coli TP giving rise to
significant sequence homology.12 The mechanism of the
phosphorolysis reaction is as follows: the ribosyl 1-ano-
meric carbon undergoes nucleophilic attack at its a face
by a phosphate ion.13,14 Norman et al. reported the crys-
tal structure of TP complexed with TPI (5-chloro-6-[1-
2. Results and discussion
Table 1 shows the effect of varying the base moiety of the
substrate in the TP-catalyzed reaction. The substrates
possessing thymine base 1 or uracil base 4 reacted easily,
and the reactivity of each substrate was approximately
the same. The thymidine of a-anomer base 5 did not
react at all. This enzyme recognized the configuration
of the 10-anomer position for the pyrimidine nucleoside
Keywords: Thymidine phosphorylase; Kinetic parameters; Substrate
recognition; Thymidine analogues.
*
Corresponding author. Tel.: +81 538 45 0177; fax: +81 538 45
0110; e-mail: a-hatano@ms.sist.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.038

A. Hatano et al. / Bioorg. Med. Chem. 16 (2008) 3866–3870
3867
Scheme 1. Phosphorolysis reaction of thymidine with thymidine phosphorylase.
Table 1. Conversions of thymidine analogues with varying the base
moiety with TP
thymine base. As the aromaticity of the pyrimidine moi-
ety increases, the leaving activity decreases, because of
increasing difficulty in polarizing the base by the amino
acids of the active site. 6-Azathymidine 8 was converted
to deoxyribose-1-phosphate in 5.4% yield during 24 h
(TP concentration was 10 U/mL). The replacement of
C-6 of this base with a nitrogen atom greatly decreased
its reactivity. The recognition of the base moiety was
very specific for thymidine phosphorylase. However,
TP has broad specificity in relation to the recognition
of the 5 position of the pyrimidine base.2 We clarified
that each of the 5-substituted uracil compounds, halo-
gen, ethyl, amino, trifluoromethyl, reacted with thymi-
dine to give the corresponding unnatural nucleoside in
good yield.
Base analogue
C10
Conversion (%)
2 h
24 h
b
86
88
0a
82
We investigated the effect of the structure of the ribosyl
ring on the TP catalyzed reaction (Table 2). Compounds
9 (b form) and 10 (a form) are 40-thionucleosides, in which
oxygen is replaced with sulfur at the ribosyl 40 position of
thymidine 1.18,19 The structure of the ribosyl ring of 40-thi-
othymidine 9 is different from native substrate 1, because
b
a
b
b
b
83
0b
Table 2. Effect of the ribosyl skeleton of the substrate on reaction with
TP
0a
0b
Thymidine analogue
Conversion (%)
86a
0a
0b
3.8b
0b
5.4a
7.9b
0b
a 10 U/mL, 24 h.
b 10 U/mL, 1 week.
0b
with high specificity. 5-Methyl-2-pyrimidinone nucleo-
side 616,17 and deoxycytidine 7 were unreactive though
the substrates have b-anomer structures. These pyrimi-
dine compounds showed lower leaving activities than
a Reaction time: 1 h, TP; 1 U/mL.
b Reaction times: 24 h, TP; 10 U/mL.

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A. Hatano et al. / Bioorg. Med. Chem. 16 (2008) 3866–3870
the atomic radius of sulfur is larger than that of oxygen.
The reactivity of compound 9 was very low, and the con-
version was 3.8% with 10 U/mL of thymidine phosphory-
lase during 24 h. Our experiment proved that TP
specifically recognizes the size of the ribosyl skeleton of
the substrate. 40-Thio-10a-thymidine 10 could not be con-
verted to the 1-phosphate form and a free thymine base.
The base of the a form did not fit in the pocket of TP as
was observed in the reaction of 5.
TP. Therefore, it can be concluded that the ribosyl ring
is necessary in the recognition of the substrate structure.
Table 3 shows the effect of different functional groups
on the ribosyl moiety on the TP catalytic reaction.
The hydroxyl groups at 30 and 50 positions were var-
iously replaced by hydrogen, amino, thiol, halide or
azide groups. The replacement of the 30 and 50 groups
decreased the reaction rate and the percentage conver-
sion with TP. The replacement of 30-OH group had a
profound effect on the reactivity; reaction of 30-deoxy-
thymidine 1523 proceeded with an initial rate of
0.74 lM/min and went to 3.2% conversion. These val-
ues were lower than those of 50-deoxythymidine 14.24
Compound 14 proceeded with an initial rate of
120 lM/min and went to 68% conversion after 1 h.
In addition to these, 30b-thymidine 13, in which the
configuration of the 30 hydroxyl group is inverted rel-
ative to 1, failed to react under the same conditions.
As also demonstrated by the replacement with the
other functional groups –NH2, –SH groups (17,25,26
1927), the 30 replacement appeared to significantly
influence the reaction with TP. These results prove
that the 30 hydroxyl group interacts with the amino
1-{[2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl}thymine
11 is a thymidine analogue possessing a flexible back-
bone.20,21 This compound bears a structure which is
analogous to an open ribosyl moiety, and could not con-
struct the oxonium intermediate by means of complex-
ing the enzyme. TP could not convert compound 11
into thymine base 3 and the corresponding phosphate
form. In compound 12 the ribosyl skeleton was replaced
with a hexopyranosyl ring.22 It was found that although
compound 12 was capable of forming an oxonium inter-
mediate, complexation with TP failed. This was most
likely due to the backbone of 12 being larger than that
of native substrate 1, giving rise to conformational dif-
ferences between this analogue and the active site of
Table 3. Conversions and initial velocities of thymidine analogues with varying functional groups on the ribosyl moiety with TP
Substrate
Conversion (%)
v0 (lM minꢀ1
)
Substrate
Conversion (%)
1 h 24 h
v0 (lM minꢀ1
)
1 h
24 h
86
91
1000
0
0
—
68
11
39
24
0
91
36
90
70
0
120
4.9
11
3.2
0.76
0
6.1
1.5
0
0.74
0.013
—
5.5
—
56
81
12.3
0
0
—

A. Hatano et al. / Bioorg. Med. Chem. 16 (2008) 3866–3870
3869
Table 4. Kinetic parameters for thymidine analogues varying the
ribosyl hydroxyl groups with thymidine phosphorylase
the leaving group. The 6-azathymidine, in which C-6
was replaced by a nitrogen atom, converted very slowly
to the corresponding nucleoside. It was found that the
leaving ability of the base moiety was lowered by an in-
crease in aromaticity of the pyrimidine ring. Phosphoryla-
tion of ribosyl 10 position was drastically effected in the
absence of the 30a-hydroxyl group, because it was neces-
sary for the enzyme to be able to bind the substrate for
nucleophilic attack. It is obvious that the 50-OH group
does not interact with the active site of the enzyme. The
ribosyl skeleton also effected the reactivity with TP. 40b-
Thiothymidine 9reacted slowly withTP, because the ribo-
syl ring was much wider than that of the native substrate.
The hexopyranosyl compound 12 does not fit in the active
site of the enzyme, as the pyranosyl skeleton is larger than
that of thymidine. The ribosyl skeleton was shown to be
very important for the recognition of the substrate by thy-
midine phosphorylase. The kinetic assay clearly showed
that a 30-OH group on the ribosyl moiety was indispens-
able for binding the substrate of TP.
Thymidine
analogue
R3
R5
Km
Vmax
(mM)
(mM/min)
1
–OH
–OH
–H
–OH
–H
2.5
10
5.2
3.4
14
15
–OH
32
0.014
acid residues of TP, and selectively binds the sub-
strate. The analogues (16,26 18,28 2024) in which the
50 group is substituted had less influence on the final
conversion, however, the initial rates decreased in
comparison with that of the native substrate 1. Com-
pounds 2224 and 23,26 with their 50 positions modified
with an iodide or azide group, respectively, were insol-
uble in phosphate buffer (pH 6.8) at 35 ꢁC, and these
substrates were not converted to the 1-phosphate form
with TP. A 20 hydroxyl group (uridine 21) resulted in
higher conversion and lower initial velocity in compar-
ison with thymidine 1. Therefore, it can be concluded
that the 30a-hydroxyl group is very important for the
recognition of the ribosyl substrate with thymidine
phosphorylase.
4. Experimental
4.1. General
The synthesis and characterization of thymidine ana-
logues used in the experiments have been previously re-
ported: 6,16,17 9 and 10,18,19 11,20,21 12,22 14,24 15,23 16,26
17,25,26 18,28 19,27 20,24 22,24 23.26 All solvents and re-
agents were of reagent-grade quality, and used without
further purification. All thymidine analogues were char-
acterized using 1H NMR, 13C NMR, EIMS. Thymidine
phosphorylase (from E. coli, EC 2.4.2.4) was purchased
from Sigma–Aldrich Chemical Co.
We performed the kinetic assay of the reaction of
thymidine analogues catalyzed by TP. Phosphate ion
concentration was kept constant at 50 mM of phos-
phate buffer (pH 6.8), and we examined the effect of
the position of the hydroxyl group on the ribosyl moi-
ety on the TP catalyzed reaction. The substrates were
thymidine 1 and the 30- and 50-deoxythymidine (14
and 15). Table 4 clearly indicates that thymidine 1
was the most effective among these substrates, Km
was 2.5 mM, and Vmax was 5.2 mM/min. Km and Vmax
for 50-deoxythymidine 14 were 4 times higher and 0.65
times lower than those of native substrate 1, respec-
tively, indicating its unsuitability as a substrate. In con-
trast, use of 30-deoxythymidine 15 in which 30-OH was
replaced by hydrogen H, had a profound effect on the
values of Km and Vmax. Vmax decreased 370 times on
replacing the 30 hydroxyl group of thymidine 1 while
the Km value of 15 increased 13-fold in comparison
to substrate 1. Thus, it can be concluded that the
30a-hydroxyl group is very important for the recogni-
tion of the ribosyl substrate. The substitution of the
ribosyl 50-OH only slightly retarded the catalytic reac-
tion with TP.
4.2. HPLC
HPLC system was a GULLIVER model by JASCO.
Each conversion was assayed with a C-18 column
(250 · 4.6 mm, Shiseido AQ or GL Science Inertsil
ODS-3) HPLC at a flow rate of 1.0 mL minꢀ1. The mo-
bile phase was 0.5–10% acetonitrile in 100 mM phos-
phate buffer (pH 6.8). The UV detector was set at
260 nm and the column was operated at 40 ꢁC.
4.3. Phosphorylation of thymidine analogues
Incubations were carried out on a small scale and gener-
ally contained 1 mM of the thymidine analogue, 1 U/mL
of thymidine phosphorylase in 1 mL of 25 mM phos-
phate buffer (pH 6.8). Mixtures were stirred at 35 ꢁC
for 1 or 24 h. The detection was by UV absorption at
260 nm of thymine base produced by thymidine.
4.4. Kinetic assays
3. Conclusion
Kinetic assay was carried out on the basis of the
Michaelis–Menten method, v0 = Vmax [S]/(Km + [S]). Ini-
tial rate v0 of each reaction was evaluated against 1 U/
mL of TP over a range of concentrations with a variety
of thymidine analogues at 35 ꢁC. The phosphate ion
concentration was kept constant at 50 mM. The data
Our results led us to conclude that not only the base moi-
ety but also the ribosyl moiety was important for the sub-
strate recognition of thymidine with thymidine
phosphorylase. It is acceptable to vary the group at the
5 position of the thymine base, however, substitution at
the 6 position of the pyrimidine has a profound effect on

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A. Hatano et al. / Bioorg. Med. Chem. 16 (2008) 3866–3870
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This research was supported partly by the Astellas
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2008.
01.038.
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