Aryl-containing esters of triphosphoric acid as substrates of terminal deoxynucleotidyl transferase

Article abstract of DOI:10.1080/15257770701490480

A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized. Copyright Taylor & Francis Group, LLC.

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Aryl-Containing Esters Of Triphosphoric
Acid As Substrates Of Terminal
Deoxynucleotidyl Transferase
A. L. Khandazhinskaya a , E. S. Matyugina a , L. A. Alexandrova a , E.
A. Shirokova a , M. K. Kukhanova a & M. V. Jasko a
a Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences , Moscow, Russia
Published online: 10 Dec 2007.
To cite this article: A. L. Khandazhinskaya , E. S. Matyugina , L. A. Alexandrova , E. A. Shirokova ,
M. K. Kukhanova & M. V. Jasko (2007) Aryl-Containing Esters Of Triphosphoric Acid As Substrates Of
Terminal Deoxynucleotidyl Transferase, Nucleosides, Nucleotides and Nucleic Acids, 26:6-7, 641-644,
DOI: 10.1080/15257770701490480
To link to this article: http://dx.doi.org/10.1080/15257770701490480
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Nucleosides, Nucleotides, and Nucleic Acids, 26:641–644, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490480
ARYL-CONTAINING ESTERS OF TRIPHOSPHORIC ACID AS
SUBSTRATES OF TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
A. L. Khandazhinskaya, E. S. Matyugina, L. A. Alexandrova, E. A. Shirokova,
M. K. Kukhanova, and M. V. Jasko
Russian Academy of Sciences, Moscow, Russia
Engelhardt Institute of Molecular Biology,
2
A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside
2
triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside
residues was synthesized.
Keywords Non-nucleoside triphosphates; terminal deoxynucleotidyltransferase
INTRODUCTION
Terminal deoxynucleotidyltransferase (TDT, EC 2.7.7.31) is a unique
template-independent DNA polymerase. TDT belongs to the X polymerase
family, a subclass of an ancient nucleotidyltransferase superfamily, which
includes nucleic acid polymerases such as DNA polymerases β, λ, µ, and
some others. Unlike any other DNA polymerases, TDT incorporates both
ribo- and deoxyribonucleotides in vitro with the equal efficacy as well as a
large array of unnatural nucleoside triphosphates. It was shown in our lab-
oratory that thriphosphate analogues bearing different bulky alkyl and aryl
groups instead of a nucleoside residue can serve as substrates for calf thy-
mus TDT, thus, demonstrating that the presence of a nucleic base in the
substrate molecule is not a determining factor for the binding to the en-
zyme active site and incorporation into the growing DNA chain.[1−3] The
efficacy of these compounds depends on substituent and linker structures
and length.[4,5] It was shown that the affinities of some of the compounds of
this series towards TDT were similar to those of natural substrates. Moreover,
non-nucleoside triphosphates (NNTP) were demonstrated to be inhibitors
and/or substrate terminators of other polymerases of the X family, particu-
larly, of human β and λ polymerases.[6]
The work was supported by the Russian Foundation for Basic Research, project No. 06-04-48248.
Address correspondence to A.L. Khandazhinskaya, Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, 32 Vavilov str., Moscow, 119991 Russia. E-mail: lhba-imb@mail.ru
641

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A. L. Khandazhinskaya et al.
RESULTS AND DISCUSSION
We present in this work the synthesis of new NNTPs of the following
structures:
Compounds (IIIa, IIIc, IVa, and IVc) were obtained by the reaction of
the corresponding aminoalcohol with 1,3-difluoro-4,6-dinitrobenzene (for
IIIa and IVa) or 1-fluoro-2,4-dinitrobenzene (for IIIc and IVc). For the
preparation of the imidazole derivative (IIIb and IVb), the corresponding
fluoro-containing precursors were treated with a solution of imidazole in
DMF. The Ludwig triphosphorylation[7] carried out without the isolation of
intermediate monophosphates gave the target triphosphates (Scheme).
The synthesized fluoro-containing triphosphates proved to be rather
labile under the conditions of ion-exchange chromatography on DEAE
(HCO−3 -form), and, therefore, were isolated in the following way. The re-
action mixture was diluted with water and passed through a Dowex 50(H+)

New Substrates of TDT
643
column. The resulting solution was concentrated, and the target product
was purified on a reversed-phase LiChroprep RP-18 column. Total yields of
the synthesized triphosphates achieved 15–30% from the starting amino al-
cohol.
All the synthesized compounds demonstrated potent substrate proper-
ties in cell-free experiments with TDT.[8]
EXPERIMENTAL SECTION
N-(2,4-Dinitro-5-fluorophenyl)-2-aminoethyl triphosphate Ia. Yield 21%.
UV (H2O, pH 6): λmax 265 nm (ε 9000), 349 nm (ε 15900). 1H-NMR (D2O):
9.10 (1H, d, J 8.1, H3), 7.04 (1H, d, J 14.6, H6), 4.22 (2H, dt, CH2), 3.75
(2H, t, J 5.3, CH2N). 31P-NMR (D2): -5.72 (1P, d, J 20.3, Pγ ), -10.39 (1P, d, J
19.3, Pα), -21.50 (1P, dd, Pβ). Mass (m/e): 484.1 [M+-1].
N-(2,4-Dinitro-5-imidazolylphenyl)-2-aminoethyl triphosphate Ib. Yield
1
17%. UV (H2O, pH 6): λmax 272 nm (ε 5800), 365 nm (ε 7000) H-NMR
(D2O): 9.24 (1H, s, H3), 8.96, 7.67 and 7.55 (3H, 3 br.s, Im), 7.50 (1H, s,
H6), 4.40 (2H, m, CH2), 3.83 (2H, t, J 5.3, CH2N). 31P-NMR (D2): -10.24
(1P, d, J 19.3, Pγ ), -10.83 (1P, d, J 20.3, Pα), -22.61 (1P, dd, Pβ). Mass (m/e):
532.2 [M+-1].
N-(2,4-Dinitrophenyl)-2-aminoethyl triphosphate Ic. Yield 24%. UV-VIS
1
(H2O, pH 6): λmax 265 nm (ε 8300), 363 nm (ε 17500). H-NMR (D2O):
8.95 (1H, d, J 2.5, H3), 8.23 (3H, dd, -5), 7.21 (1H, d,J 9.65, H6), 4.01 (2H,
m, CH2), 3.72 (2H, t, J 5.9, CH2N).31P NMR (D2O): δ -9.88 (d, 1P, J 21.4,
Pγ ), -10.32 (d, 1P, J 19.3, Pα), -21.77 (dd, 1P, Pβ). Mass (m/e): 466.1 [M+-1].
N-(2,4-Dinitro-5-fluorophenyl)-4-aminobutyl triphosphate IIa. Yield
15%. UV (H2O, pH 6): λmax 265 nm (ε 9100), 349 nm (ε 16000).1H-NMR
(D2O): 9.10 (1H, d, J 8.1, H3), 6.94 (1H, d, J 15.6, H6), 4.01 (2H, m, CH2),
3.50 (2H, t, J 6.8, CH2N), 1.77 (4, m, (CH2)2). 31P-NMR (D2): -10.12 (1P, d,
J 19.3, Pγ ), -10.32 (1P, d, J 20.3, Pα), -22.65 (1P, dd, Pβ). Mass (m/e): 512.1
[M+-1].
N-(2,4-Dinitro-5-imidazolylphenyl)-4-aminobutyl triphosphate IIb. Yield
23%. UV (H2O, pH 6): λmax 272 nm (ε 5800), 365 nm (ε 7000).1H-NMR
(D2O): 9.19 (1H, s, H3), 8.30, 7.45 and 7.33 (3H, 3 br.s, Im), 7.19 (1H, s,
H6), 3.99 (2H, m, CH2), 3.54 (2H, t, J 6.8, CH2N), 1.82-1.72 (4, m, (CH2)2).
31P-NMR (D2): -10.10 (1P, d, J 19.3, Pγ ), -10.31 (1P, d, J 20.3, Pα), -22.64 (1P,
dd, Pβ). Mass (m/e): 560.2 [M+-1].
N-(2,4-Dinitrophenyl)-4-aminobutyl triphosphate IIc. Yield 30%. UV-VIS
1
(H2O, pH 6): λmax 265 nm (ε 8300), 363 nm (ε 17500). H-NMR (D2O):
9.19 (1H, d, J 2.8, H3), 8.30 (3H, dd, -5), 7.19 (1H, d, J 9.65, H6), 3.99 (2H,
m, CH2), 3.54 (2H, t, J 6.8, CH2N), 1.82-1.72 (4, m, (CH2)2). 31P-NMR (D2):
-10.10 (1P, d, J 19.3, Pγ ), -10.31 (1P, d, J 20.3, Pα), -22.64 (1P, dd, Pβ). Mass
(m/e): 494.2 [M+-1].

644
A. L. Khandazhinskaya et al.
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