Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis

Article abstract of DOI:10.3762/bjoc.14.289

Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-l-AMP) using TthAPRT and 1-(β-Dribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthPRT.

Full text of DOI:10.3762/bjoc.14.289

Thermophilic phosphoribosyltransferases
Thermus thermophilus HB27 in nucleotide synthesis
Ilja V. Fateev, Ekaterina V. Sinitsina, Aiguzel U. Bikanasova, Maria A. Kostromina,
Elena S. Tuzova, Larisa V. Esipova, Tatiana I. Muravyova, Alexei L. Kayushin,
Irina D. Konstantinova* and Roman S. Esipov*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 3098–3105.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia
doi:10.3762/bjoc.14.289
Received: 05 July 2018
Email:
Accepted: 27 November 2018
Published: 21 December 2018
Irina D. Konstantinova* - kid1968@yandex.ru; Roman S. Esipov* -
esipov@ibch.ru
This article is part of the thematic issue "Enzymes in chemical
transformations".
* Corresponding author
Keywords:
Guest Editor: K. N. Allen
adenine phosphoribosyltransferase; catalysis; enzyme; hypoxanthine
phosphoribosyltransferase; multi-enzyme cascade; nucleotides;
thermophiles
© 2018 Fateev et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The
recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus
thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10–13 mg per liter of
culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate speci-
ficity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two
nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-Сl-AMP) using TthAPRT and 1-(β-D-
ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthНPRT.
Introduction
Bacterial phosphoribosyltransferases are used in multi-enzy- corresponding nucleotides (Figure 1). The use of thermophilic
matic cascades that perform nucleotide synthesis de novo [1,2]. phosphoribosyltransferases allows carrying out reactions at a
Recently, we reported on the possibility of cascade synthesis, higher temperature, so the concentrations of heterocyclic bases
where enzymes of thermophilic microorganisms Thermus ther- can be increased [1-3].
mophilus HB27 (phosphoribosylpyrophosphate synthetase –
PRPPS and adenine phosphoribosyltransferase – APRT) and There is great interest in the development of multi-enzymatic
Thermus sp. 2.9 (ribokinase – RK) carry out successive trans- cascades [4-9] for the preparation of nucleosides and
formations of ribose and adenine heterocyclic bases into the nucleotides due to the regio- and stereospecificity of enzymes
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Figure 1: A multi-enzymatic synthesis of modified adenosine -5'-monophosphates.
[4,10,11], performing metabolic transformations of substrates. TthAPRT and TthHPRT was performed to determine the
Phosphoribosyltransferases are increasingly being widely used usability of thermophilic transferases in nucleotide synthesis. A
as key enzymes in multi-enzymatic systems [2]. The substrate scheme of purine nucleotide synthesis using TthAPRT and
specificity of APRT limits the number of possible nucleotides TthHPRT is shown in Figure 2.
that can be synthesized. Thus, for Thermus thermophilus HB27
APRT (TthAPRT), nucleotide synthesis is limited to the closest
structural homologs of adenine (Table 1) [1].
Table 1: Substrates for TthAPRT.
Base
Conversion into nucleotide
(24 h, %)a
2,6-diaminopurine
2-chloroadenine
2-fluoroadenine
adenine
16.80
97.58
36.50
50.02
60.88
78.2
2-methoxyadenine
N1-methyladenine
N6-benzyladenine
2-aminobenzimidazole
1.87
0.09
aReaction mixtures (0.5 mL, 20 mM Tris-HCl, pH 8.0, 75 °C) contained
0.4 mM heterocyclic base, 0.4 mM PRPP, 0–5 mM MgCl2, 1.25 μg
TthAPRT [1].
Figure 2: Nucleotide synthesis using phosphoribosyltransferases.
Unfortunately, 1,2,4-triazole-3-carboxamide, its analogues, Results and Discussion
guanine, hypoxanthine, and 7-deazapurins are not substrates for Genes TT_RS08985 and TT_RS06315 from T. thermophilus
TthAPRT. This severely limits the usability of multi-enzymatic HB27, coding TthHPRT and TthAPRT, were cloned into
cascades in the synthesis of nucleotides, including the modified expression plasmid vectors pET 23a+ and pET 23d+, respec-
ones.
tively. The resulting recombinant plasmid pER-TthHPRT
contained fusion gene HPRT-HisTag coding TthHPRT with a
To expand the possible repertoire of nucleotides that could be C-terminal His-Tag. The resulting recombinant plasmid pER-
synthesized, we obtained the recombinant form of hypoxathine TthAPRT contained the gene APRT coding TthAPRT without
phosphoribosyltransferase Thermus thermophilus (TthHPRT), any additional sequences. Nucleotide sequences of the cloned
investigated its substrate specificity and optimal conditions for genes were verified by sequencing. The codone GGG→AGG
catalytic activity, and determined the kinetic parameters of the substitution corresponding to amino-acid Arg27Gly replace-
enzyme. A comparative study of the substrate specificity of ment was found in the gene encoding the TthHPRT.
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Beilstein J. Org. Chem. 2018, 14, 3098–3105.
The screening of available producer strains was performed to of 5-phosphoribosyl-α-1-pyrophosphate (1 mM), it can be
find strains, which produce target enzymes in soluble form. The assumed that the presence of magnesium ions promotes the
resulting strains E. coli BL21(DE3)/pER- TthAPRT and E. coli proper spatial orientation of the substrate. The reaction also
C3030/pER- TthHPRT produced enzymes mainly in soluble proceeds in the absence of magnesium ions in solution. A simi-
form (>80%).
lar dependence is observed for TthAPRT.
The established procedure for isolation and purification of
TthHPRT includes heat treatment, immobilized metal affinity
chromatography, final size-exclusion chromatography, and con-
centration. For TthAPRT, the protocol include heat treatment,
anion exchange chromatography, hydrophobic chromatography,
final size-exclusion chromatography, and concentration. Yields
of both transferases were no less than 10–13 mg per liter of cul-
ture, with a purity of about 96% (as determined by SDS-
PAGE).
The influence of temperature and Mg2+ concentration on the ac-
tivity of TthHPRT was investigated. The results were compared
with data for adenine phosphoribosyltransferase Thermus ther-
mophilus, obtained earlier [1].
Figure 4: Dependence of TthHPRT and TthAPRT activity on the Mg2+
concentration (reaction mixtures (0.5 mL) contained 20 mM Tris-HCl,
pH 8.0, 1 mM 5-phosphoribosyl-1-α-pyrophosphate, and 0–5 mM
MgCl2; in case of TthHPRT mixtures contained 1 mM hypoxanthine
and 0.18 μg of enzyme, in case of TthAPRT – 1 mM adenine and
0.125 μg of enzyme; reactions were performed at 70 °C; 100% – activi-
ty at 1 mM concentration).
The TthAPRT is active over a wide temperature range
(Figure 3). A maximal activity of TthHPRT (1.1 unit/mg) is ob-
served at 60 °C. The activity at 36 °C is 5% from the maximal
one and at 90 °C it is 3% from the maximal one. It is interest- After optimization of the reaction conditions, kinetic parame-
ing, that TthAPRT shows its maximal activity at 75 °C.
ters for TthHPRT were determined (Table 2).
Based on the Km values, the affinity of 5-phosphoribosyl-α-1-
pyrophosphate for the active site is much lower than that of
heterocyclic bases. The similar situation we observed for
TthAPRT [1]. Comparison of the synthesis rates of inosine-5'-
monophosphate and guanosine-5`-monophosphate showed that
the first is synthesized 4.6 times faster. The literature data for
similar enzymes (see Table 2) confirm a poor affinity of PRPP
to the active site: Km for hypoxanthine is 17 fold less then for
PRPP, although for the human enzyme Km is only 5 fold less.
Comparing two enzymes from different strains of Thermus ther-
mophilus, we can conclude that TthAPRT from HB8 (in
contrast with HB27), synthesizes guanosine-5`-monophosphate
faster. This may be due to the difference in reaction conditions.
Kinetic data are displayed by double reciprocal plot (Figure 5).
Determination of substrate specificity of TthHPRT was per-
formed in comparative experiments with TthAPRT. The process
of nucleotide synthesis was monitored by a liquid chromatogra-
phy–mass spectrometry analysis of the reaction mixture.
Figure 3: Dependence of TthHPRT and TthAPRT activity on tempera-
ture (reaction mixtures (0.5 mL) contained 20 mM Tris-HCl, pH 8.0,
1 mM 5-phosphoribosyl-1-α-pyrophosphate, and 5 mM MgCl2; in the
case of TthHPRT mixtures contained 1 mM hypoxanthine and 0.18 μg
of enzyme, in the case of TthAPRT – 1 mM adenine and 0.125 μg of
enzyme).
The influence of the magnesium ion concentration on the
TthHPRT activity is nonlinear. The activity increases rapidly
while the magnesium chloride concentration increases from 0 to
1 mM (Figure 4). Further increasing of the concentration (up to
5 mM) does not increase the activity significantly. Since the
reaction rate increases rapidly with increasing the magnesium
chloride concentration to values equivalent to the concentration
The data is presented in the Table 3. As expected, TthHPRT is
specific to 6-oxopurines, while TthAPRT is specific to
6-aminopurines. Both enzymes do not recognize thymine as a
substrate. This is consistent with data that pyrimidine hetero-
cyclic bases are substrates for uracyl phosphoribosyltransferase
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Beilstein J. Org. Chem. 2018, 14, 3098–3105.
Table 2: Kinetic parameters of inosine-5'-monophosphate and guanosine-5'-monophosphate synthesis using HPRT from various organisms.
Substrate
Km, μМ
Vmax, μmol/min·mg
kcat, 1/s
kcat/Km, 1/M·s
Thermus thermophilus HB27
hypoxanthine
guanine
13 ± 4
28 ± 9
6 ± 2
9 ± 3
6.9 × 105
7.1 × 104
2.7 × 104
28 ± 9
2.0 ± 0.7
6 ± 2
PRPP
220 ± 60
17 ± 5
Thermus thermophilus HB8 [12]
hypoxanthine
guanine
3.9 ± 1.5
–
–
–
9.1 ± 0.8
18 ± 1
–
–
–
7.4 ± 1.7
68 ± 18
PRPP
20 ± 2
Homo sapiens [13]
hypoxanthine
PRPP
3.8 ± 0.3
–
–
2.6 ± 0.6
7 × 105
1 × 105
19.1 ± 1.6
2.5 ± 0.05
Escherichia coli [14]
Hypoxanthine
PRPP
37
–
–
–
–
–
–
330
Table 3: Substrate specifity of TthHPRT and TthAPRT.
Base
Conversion (24 h, %)a
TthHPRT TthAPRT
adenine
5.3
91.0
73.9
0
48.1
6.4
25.6
52.9
31.1
4.8
57.4
1.0
0
hypoxanthine
guanine
2-chloroadenine
2-fluoroadenine
6-mercapopurine
allopurinol
0
85.1
39.3
80.6
0
8-azaguanine
thymine
1,2,4-triazole-3-carboxamide
0
0
Figure 5: Lineweaver–Burk plot for synthesis of inosine-5'-monophos-
phate and guanosine-5'-monophosphate.
1,2,4-triazole-3-carboxy-N-methyl-
amide
0
0
aReaction mixtures (0.125 mL, 20 mM Tris-HCl, pH 8.0, 60 °C)
contained 0.5 mM heterocyclic base, 0.5 mM PRPP, 0.5 mM MgCl2,
5 mМ KH2PO4 and 0.4 μg of TthHPRT or TthAPRT.
and orotate phosphoribosyltransferase only [2]. Unfortunately,
we did not find any product in reactions with compounds based
on 1,2,4-triazole-3-carboxamide, which was also observed for
E. сoli HPRT [15,16]. However, allopurinol and 8-azaguanine
are substrates for TthHPRT, and 2-chloroadenine is a substrate
for TthAPRT. For 2-chloroadenine and 8-azaguanine, reaction
at a higher temperature is preferable because of their low solu-
bility in water (less than 1 mM at 37 °C). Interestingly, allo-
purinol proved to be a good substrate for both TthHPRT and
TthAPRT, unlike hypoxanthine, which differs only in the posi-
tion of one of the nitrogen atoms. Probably, the presence of
nitrogen atom at C7 position of purine heterocycles plays an im-
portant role in reactions catalyzed by phosphoribosyltrans-
ferase, and also affects the substrate properties of TthHPRT and
TthAPRT.
Two nucleotides were synthesized using TthHPRT or TthAPRT
(see Figure 6). Synthesis of 2-Cl-AMP was performed at 75 °C.
This allowed to achieve a concentration of 0.5 mM of the initial
2-chloroadenine. The reaction progress was monitored by
HPLC. After 2 days (the product content in the reaction mix-
ture was 54%), the reaction mixture was concentrated and the
desired product was isolated by column chromatography on ion-
exchange sorbents (anion and then cation-exchange). The yield
of 2-Сl-AMP was 37%. A second nucleotide (Allop-MP) was
synthesized at a lower temperature (60 °C). After 2 days, the
product content in the reaction mixture was 55%. The product
was isolated in the same way, with a yield of 32%.
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Beilstein J. Org. Chem. 2018, 14, 3098–3105.
Becton Dickinson Biosciences (USA, NJ). NaOH and HCl were
purchased from Merck (USA, MA). Sodium persulfate,
TEMED, ethidium bromide, and sodium azide were purchased
from Helicon (Russia). dNTP was purchased from Thermo
Fisher Scientific (USA, MA). DTT, phenylmethylsulfonyl chlo-
ride, magnesium chloride, nickel sulfate, potassium dihy-
droorthophosphate, Ni-IDA sepharose, 5-phosphoribosyl-α-1-
pyrophosphate and all bases (adenine, hypoxanthine, guanine,
2-chloroadenine, etc.) were purchased from Sigma-Aldrich
(USA, MO).
Bacterial strains: a) E. coli C3030 [MiniF lysY (CamR) / fhuA2
lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC
(SpecR, lacIq) ΔtrxB sulA11 R(mcr-73::miniTn10--TetS)2 [dcm]
R(zgb-210::Tn10 --TetS) endA1 Δgor ∆(mcrC-mrr)114::IS10]
New England Biolabs (USA, MA), b) E.coli BL21(DE3) fhuA2
[lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo
ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5.
Figure 6: Synthesis of nucleotides 2Cl-AMP and Allop-MP using phos-
phoribosyltransferases TthAPRT or TthHPRT respectively.
Plasmid vector: pET 23a+, pET 23d+ (Merck Millipore, USA,
MA).
Conclusion
Enzymes: NdeI, XhoI, NcoI, T4 DNA-ligase (Thermo Scien-
The recombinant adenine phosphoribosyltransferase and hypox- tific, USA, MA), Encyclo-polymerase (Eurogen, Russia).
anthine phosphoribosyltransferase from Thermus thermophilus
HB27 were purificated with yields no less than 10–13 mg per The protein concentration was determined by the Bradford
litre of culture. A comparative study of substrate specificity of method [17], using BSA as a standard.
these enzymes towards different heterocyclic bases was carried
out and temperature-dependence and magnesium chloride con- Protein purity was determined by electrophoresis in a polyacryl-
centration-dependence of enzymes activity were determined. amide gel under denaturing conditions [18].
TthHPRT can be used for the synthesis of nucleotides contain-
ing different purine derivatives including 8-aza- and 8-aza-7- Cloning and creation of producer strain: Genes
deazapurine. The use of hypoxanthine and adenine transferases TT_RS08985 and TT_RS06315, encoding TthHPRT and
in multi-enzyme cascades significantly extends the spectrum of TthAPRT, respectively, were amplified on the genomic DNA
synthetic purine nucleotides. Two nucleotides were synthesized: template of the T. thermophilus HB27 strain by a polymerase
9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate chain reaction (PCR) using synthetic primers. The genes were
(2-Сl-AMP) using TthAPRT and 1-(β-D-ribofuranosyl)pyra- cloned into the expression vectors pET-23a+ and pET-23d+ re-
zolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) spectively. The E. coli strains BL21(DE3)/pER- TthAPRT and
using TthНPRT with yields of 37% and 32%, respectively. C3030/pER- TthHPRT produced the target enzymes mainly in
Using of hypoxanthine and adenine transferases in multi-en- soluble form (culturing conditions: 4 h grow at 37 °С after
zyme cascades significantly extends the spectrum of synthetic supplementing with 0.4 mM IPTG).
purine nucleotides.
Isolation and purification of TthHPRT: A cell pellet was
Experimental
resuspended in 50 mM Tris-HCl, 200 mМ NaCl, and 1 mМ
Tris-buffer, acetic acid, sodium chloride, glycerol, acrylamide, phenylmethylsulfonyl fluoride (PMSF) рН 8.0 (1:10 w/v). The
N,N'-bisacrylamide, ATP, bromophenol blue, agarose, EDTA, cells were disrupted by sonication for 30 min at 20 kHz at
IPTG, ampicillin, sodium dodecylsulfate, imidazole and DMF +4 °С. The cell debris was pelleted by centrifugation at
were purchased from Panreac (Spain, Barselona). Ethanol was 12,000 rpm for 30 min at +4 °С. The cell lysate was heat-treated
purchased from MedChemProm. Coomassie Brilliant Blue (65 °С, 10 min) and the pellet was removed by centrifugation.
R-250 was purchased from Bio-Rad (USA, CA). Bacto yeast The supernatant was applied to a column XK 16/20 (GE Health-
extract, bacto tryptone, and bacto agar were purchased from care, USA) packed with Ni-IDA Sepharose (Sigma Aldrich,
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Beilstein J. Org. Chem. 2018, 14, 3098–3105.
USA) pre-equilibrated with 50 mM Tris-HCl and 200 mM NaCl Product quantities were determined as described in the "En-
at pH 8.0. Ballast proteins were eluted with a solution, contain- zyme assay" section. Each experiment was repeated three times.
ing 50 mM Tris-HCl, 50 mM imidazole, and 200 mM NaCl, Kinetic parameters were determined by nonlinear regression
pH 8.0 (4 CV, flow rate 2 mL/min). The target enzyme was analysis using SciDAVis v0.2.4 software (free software, web
eluted with solution, contained 50 mM Tris-HCl, 250 mM site: scidavis.sourceforge.net). Catalytic constants (kcat) were
imidazole, and 200 mM NaCl, pH 8.0 (4 CV, flow rate calculated per 1 subunit (20.3 kDa, calculated based on amino
2 mL/min). Pooled fractions were concentrated by a polysul- acid sequence).
fonic membrane PBGC 10 kDa (Millipore, USA). The result-
ing solution was applied to a column with HiLoad 16/60 Mass spectra were measured on an Agilent 6224, ESI-TOF,
Superdex 75pg (GE Healthcare, USA), equilibrated by 20 mM LC/MS (USA) in positive ion mode (ESI), LCQ Fleet ion trap
Tris-HCl, 50 mM NaCl, 0.04% NaN3, and 10% glycerol, mass spectrometer (Thermo Electron, USA) and Agilent 1100
pH 8.0. Fractions, containing the target enzyme with a purity of LC/MSD VL (Agilent Technologies) equipped an APCI and
more than 96 %, were pooled and concentrated up to a concen- ESI source (positive and negative mode of ionization), 1100
tration of 13 ± 1 mg/mL.
DAD and ELSD PL-ELS 1000 (Polymer Laboratories).
Isolation and purification of TthAPRT: Cell biomass disrup- Nucleotides synthesis
tion and heat-treatment was performed as described in section 9-(β-D-Ribofuranosyl)-2-chloroadenine 5'-monophosphate
"Isolation and purification of TthHPRT". The resulting solution (2-Сl-AMP): 2-Chloroadenine (17 mg, 0.10 mmol) was dis-
was diluted (2-fold) with solution, comtained 50 mM Tris-HCl, solved in water (203 mL) under stirring and heating at 90 °C ,
2 M (NH4)2SO4, pH 8.0, and applied to column XK 16/20 and after cooling to 70 °C, magnesium chloride hexahydrate
packed with Phenyl Sepharose HP (GE Healthcare, USA). The (41 mg, 0.21 mmol) and potassium dihydroorthophosphate
column was eluted by linear gradient of (NH4)2SO4 (1.0 – 0 M, (276 mg, 2.03 mmol) were added. The pH of the solution was
12 CV, flow rate 2 mL/min). Fractions, contained the target en- adjusted to 8.0 by 2 N potassium hydroxide. The pentasodium
zyme, were pooled and concentrated on polysulphonic mem- salt of 5-phosphoribosyl-α-1-pyrophosphate (70 mg,
brane PBGC 10 kDa. The resulting solution was applied to 0.14 mmol) and TthAPRT (5 units) were added, and the reac-
column with HiLoad 16/60 Superdex 200, equilibrated by tion mixture was incubated at 75 °C for 2 days; the reaction
20 mM Tris-HCl, 50 mM NaCl, 0.04% NaN3, and 5% glycerol, progress was monitored by HPLC. The reaction mixture was
pH 8.0. Fractions, contained the target enzyme with purity more neutralized with 2 N hydrochloric acid and concentrated in
than 96%, were pooled and concentrated up to concentration vacuo to ca. 10 mL. The precipitate was filtered off, the filtrate
12 ± 1 mg/mL.
was applied to the column with DEAE-Toyopearl 650C, bicar-
bonate form, 40 × 140 mm, and the product was eluted with tri-
Enzyme assay: Each reaction mixture (0.5 mL, 20 mM Tris- ethylammonium bicarbonate (0.1 M). Fractions were concen-
HCl, pH 8.0) contained 1 mM 5-phosphoribosyl-1-α-pyrophos- trated in vacuo to ca. 10 mL, applied to the column with
phate, 1 mM hypoxanthine, 5 mM MgCl2, and hypoxanthine CM-Sephadex C-25, sodium form, 20 × 160 mm, and the prod-
phosphoribosyltransferase Thermus thermophilus (0.18 μg). uct was eluted with water to give, after evaporation and drying
Reaction mixtures were incubated at 70 °C. Substrate and prod- in vacuo under P2O5, 16 mg (0.037 mmol; 37%) of 9-(β-D-ribo-
uct quantities were determined using HPLC (Waters 1525, furanosyl)-2-chloroadenine 5'-monophosphate of 99% purity
column Ascentis Express C18, 2.7 μm, 3.0 × 75 mm, eluent A (HPLC). HRMS (ESI+): m/z [M + H]+ calcd for
0.1% aqeous TFA, eluent B 0.1% TFA / 70% acetonitrile in C10H13N5O7P1Cl1: 382.0315; found, 382.0353; [2M + H]+,
water, detection at 254 nm, Waters 2489).
found, 763.0606; [Base + H]+, found, 170.0244; 1Н NMR
(DMSО-d6) δ ppm) 8.52 (s, 1Н, H8), 7.78 (br. s., 2H, NH2),
Kinetic parameters determination: Each reaction mixture 5.83 (d, J1’,2’ = 6 Hz, 1Н, H1’), 4.61 (m, 1Н, H2’), 4.23 (m, 1Н,
(1.0 mL, 20 mM Tris-HCl, pH 8.0) contained 5 mM MgCl2, H3’), 4.06 (m, 1Н, H4’), 3.84 (m, 2Н, H5a’, H5b’) ppm;
hypoxanthine phosphoribosyltransferase Thermus thermophilus 13C NMR (DMSО-d6) δ 156.61 (C2 or C6), 153.01 (C6 or
(0.18 μg), and the following components: a) hypoxanthine C2), 150.73 (C4), 139.68 (C8), 117.57 (C5), 86.40 (C1’),
(0.01–0.50 mM) or guanine (0.01–0.20 mM) and 1 mM 5-phos- 84.52 (C4’), 74.13 (C2’), 71.06 (C3’), 3.94 (C5’) ppm;
phoribosyl-1-α-pyrophosphate to determine Km and Vmax for 15N NMR (DMSО-d6) δ 242.7 (N7), 171.3 (N9), 86.84 (NH2)
hypoxanthine and guanine, and b) 5-phosphoribosyl-1-α- ppm.
pyrophosphate (0.05–1.20 mM) and 0.50 mM hypoxanthine to
determine Km and Vmax for 5-phosphoribosyl-1-α-pyrophos- 1-(β-D-Ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one
phate. Reaction mixtures were incubated at 70 °C for 2 min. 5'-monophosphate (Allop-MP): Allopurinol (14 mg,
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Beilstein J. Org. Chem. 2018, 14, 3098–3105.
ORCID® iDs
0.10 mmol) was dissolved in water (203 mL) under stirring and
heating at 90 °C, and after cooling to 50 °C, magnesium chlo-
ride hexahydrate (41 mg, 0.21 mmol) and potassium dihy-
droorthophosphate (276 mg, 2.03 mmol) were added. The pH of
the solution was adjusted to 8.0 with 2 N potassium hydroxide.
The pentasodium salt of 5-phosphoribosyl-α-1-pyrophosphate
(70 mg, 0.14 mmol) and TthHPRT (5 units) were added, and the
reaction mixture was incubated at 60 °C for 2 days; the reaction
progress was monitored by HPLC. The reaction mixture was
neutralized by 2 N hydrochloric acid and concentrated in vacuo
to ca. 10 mL. The precipitate was filtered off, the filtrate was
placed on the column with DEAE-Toyopearl 650C, bicar-
bonate form, 40 × 140 mm, and the product was eluted with tri-
ethylammonium bicarbonate (0.2 M). Fractions were concen-
trated in vacuo to ca. 10 mL, applied to the column with
CM-Sephadex C-25, sodium form, 20 × 160 mm, and the prod-
uct was eluted with water to give, after evaporation and drying
in vacuo under P2O5, 11 mg (0.032 mmol; 32%) of 1-(β-D-ribo-
furanosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate
of 97% purity (HPLC). HRMS (ESI+): m/z [M + H]+ calcd for
C10H13N4O8P1: 349.0545; found, 349.0520; [2M + H]+, found,
697.0952; [3M + H]+, found, 1045.1374; [Base + H]+ found,
137.0453; 1Н NMR (DMSО-d6) δ 12.44 (br. s, 1H, NH), 8.15
(s, 1H, H3), 8.13 (s, 1H, H6), 6.06 (d, J = 4.1 Hz, 1H, H1’),
4.56 (dt, 1H, H2’, J = 4.54; <0.5), 4.31 (t, 1H, H3’, J = 4.8),
4.04 (m, 1H, H4’), 3.85 (ddd, J = 11.0, 7.6; 6.2 Hz, 1H, H5’a),
3.66 (ddd, J = 11.0, 7.2, 6.1 Hz 1H, H5’b) ppm; 13C NMR
(DMSО-d6) δ 157.03 (C4), 152.90 (C7a), 148.53 (C6), 135.38
(C3), 106.06 (C4a), 88.16 (C1’), 83.27 (C4’), 73.39 (C2’),
71.38 (C3’), 64.76 (C5’) ppm; 15N NMR (DMSО-d6) δ 302.8
(N2), 210.6 (N7), 204.9 (N1), 171.1 (N5).
Ilja V. Fateev - https://orcid.org/0000-0002-8571-4197
Irina D. Konstantinova - https://orcid.org/0000-0001-5563-6549
References
1. Esipov, R. S.; Abramchik, Yu. A.; Fateev, I. V.; Konstantinova, I. D.;
Kostromina, M. A.; Muravyova, T. I.; Artemova, K. G.; Miroshnikov, A. I.
ActaNaturae 2016, 8, 82–90.
2. Del Arco, J.; Fernández-Lucas, J. Curr. Pharm. Des. 2017, 23, 1–15.
doi:10.2174/1381612823666171017165707
3. Taran, S. A.; Verevkina, K. N.; Feofanov, S. A.; Miroshnikov, A. I.
Russ. J. Bioorg. Chem. 2009, 35, 739–745.
doi:10.1134/s1068162009060107
4. Mikhailopulo, I. A. Curr. Org. Chem. 2007, 11, 317–335.
doi:10.2174/138527207780059330
5. Mikhailopulo, I. A.; Miroshnikov, A. I. Mendeleev Commun. 2011, 21,
57–68. doi:10.1016/j.mencom.2011.03.001
6. Wintersberger, E. Biochem. Soc. Trans. 1997, 25, 303–308.
doi:10.1042/bst0250303
7. Tesmer, J. J.; Klem, T. J.; Deras, M. L.; Davisson, V. J.; Smith, J. L.
Nat. Struct. Biol. 1996, 3, 74–86. doi:10.1038/nsb0196-74
8. Kim, M.-J.; Whitesides, G. M. Appl. Biochem. Biotechnol. 1987, 16,
95–108. doi:10.1007/bf02798359
9. Scism, R. A.; Stec, D. F.; Bachmann, B. O. Org. Lett. 2007, 9,
4179–4182. doi:10.1021/ol7016802
10.Mikhailopulo, I. A.; Miroshnikov, A. I. ActaNaturae 2010, 2, 36–59.
11.Iglesias, L. E.; Lewkowicz, E. S.; Medici, R.; Bianchi, P.; Iribarren, A. M.
Biotechnol. Adv. 2015, 33, 412–434.
doi:10.1016/j.biotechadv.2015.03.009
12.Kanagawa, M.; Baba, S.; Ebihara, A.; Shinkai, A.; Hirotsu, K.;
Mega, R.; Kim, K.; Kuramitsu, S.; Sampei, G.-i.; Kawai, G.
Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2010, 66,
893–898. doi:10.1107/s1744309110023079
13.Canyuk, B.; E-Wan, A.; Keawwijit, W.; Nualnoi, T.; Sirisatean, L.;
Tansakul, P.; Tanthana, C. Nucleosides, Nucleotides Nucleic Acids
2008, 27, 894–899. doi:10.1080/15257770802146593
14.Miller, R. L.; Ramsey, G. A.; Krenitsky, T. A.; Elion, G. B. Biochemistry
1972, 11, 4723–4731. doi:10.1021/bi00775a014
Abbreviations
15.Streeter, D. G.; Miller, J. P.; Robins, R. K.; Simon, L. N.
Ann. N. Y. Acad. Sci. 1977, 284, 201–210.
APRT – adenine phosphoribosyltransferase; HPRT –
hypoxathine phosphoribosyltransferase; PRPPS – phosphoribo-
sylpyrophosphate synthetase; RK – ribokinase; Tth - Thermus
thermophilus; 2-Сl-AMP – 9-(β-D-ribofuranosyl)-2-chloro-
adenine 5'-monophosphate; Allop-MP – 1-(β-D-ribofuranosyl)-
pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate
doi:10.1111/j.1749-6632.1977.tb21952.x
16.Naesens, L.; Guddat, L. W.; Keough, D. T.; van Kuilenburg, A. B. P.;
Meijer, J.; Voorde, J. V.; Balzarini, J. Mol. Pharmacol. 2013, 84,
615–629. doi:10.1124/mol.113.087247
17.Laemmli, U. K. Nature 1970, 227, 680–685. doi:10.1038/227680a0
18.Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
doi:10.1016/0003-2697(76)90527-3
Supporting Information
Supporting Information File 1
Detailed analysis of mass spectrometry and NMR data.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-14-289-S1.pdf]
Acknowledgements
The authors are grateful to Russian Science Foundation (proj-
ect No. 14-50-00131) for financial support of this work.
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