A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA

Article abstract of DOI:10.1016/S0960-894X(02)00650-9

An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium incorporated at the 1′, 4′, or 5′ position. Three intermediates-[1-²H]-D-ribose, [4-²H]-D-ribose, and [5-²H

Full text of DOI:10.1016/S0960-894X(02)00650-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3093–3096
A General Synthesis of Specifically Deuterated Nucleotides for
Studies of DNA and RNA
Bingzi Chen, Elizabeth R. Jamieson and Thomas D. Tullius*
Department of Chemistry, Boston University, Boston, MA 02215, USA
Received 19 March 2002; accepted 1 August 2002
Abstract—An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium
incorporated at the 10, 40, or 50 position. Three intermediates-[1-2H]-d-ribose, [4-2H]-d-ribose, and [5-2H2]-d-ribose-were prepared
by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic reactions. Milligram
quantities of the desired products were obtained with an average deuterium content of 96ꢀ1%.
# 2002 Elsevier Science Ltd. All rights reserved.
Specifically deuterated ribonucleotides and deoxyribo-
nucleotides have important applications in biochemistry
and molecular biology. Deuterated nucleotides can be
incorporated into DNA and RNA molecules to suppress
nonessential proton resonances in NMR structural
studies.1,2 Another use for these molecules has been in
atom transfer and kinetic isotope effect experiments with
various DNA damaging agents, such as calicheamicin,3
bleomycin4 and the hydroxyl radical.5 While these types
of experiment provide valuable information, their
application to biochemical problems has been limited
due to the difficulty in synthesizing specifically labeled
nucleotides. Here we describe a convenient scheme for
preparing specifically deuterated deoxyribonucleotides
and ribonucleotides using a combination of chemical
and enzymatic syntheses.
Using a combination of chemical and enzymatic methods
greatly simplifies the synthesis, in particular eliminating
the need for multistep chemical coupling, deoxygenation,
and phosphorylation to produce the desired (deoxy)-
nucleoside triphosphates. A further advantage of this
scheme is the ease of preparation of a large variety of
nucleotide products from a relatively small number of
chemically synthesized precursors. While this work
describes the synthesis of specifically deuterated purine
nucleotides, this procedure could easily be extended to
produce pyrimidine nucleotides.7 The nucleoside triphos-
phates made by this method are suitable for synthesizing
larger RNA and DNA molecules, as we show here by their
use in an in vitro transcription reaction to produce spe-
cifically deuterated L-21 Sca ribozyme.
We prepared [1-2H]-d-ribose 2 by reduction of d-ribo-
nolactone with sodium amalgam in D2O, with a yield of
89% (Scheme 2a). We followed the synthetic scheme
developed by Townsend and coworkers6 to prepare
ribose specifically deuterated at either the 40 or 50 position.
The syntheses of [4-2H]- and [5-2H2]-d-ribose 10 and 14
(Scheme 2b and c) began with protection of the 20 and 30
hydroxyl groups of d-ribose. After further protection of
either the 50 or 10 hydroxyl, the protected ribose was
oxidized. Reduction of the resulting ketone 7 or ester 12
by LiAlD4 introduced deuterium at either the C-4 or the
C-5 position. 1H NMR spectroscopy indicated that
deuterium was incorporated into 9 and 13, as the signal for
the 40 proton or the 50 protons completely disappeared,
compared to the corresponding non-deuterated com-
pounds. The protected deuterated ribose was hydrolyzed
Synthesis of a series of nucleotides with deuterium
incorporated at the 10, 40, or 50 position of the deoxyribose
or ribose ring was accomplished in stages (Scheme 1).
Chemical transformation of d-ribose or d-ribono-
lactone was carried out through a series of reactions6 to
incorporate the isotope at a specific position. One-pot
enzymatic phosphorylation and coupling of the deuterated
ribose sugar to a base7 allowed the direct formation of
the desired ribonucleotides. Deoxyribonucleotides were
produced by enzymatic reduction of the corresponding
ribonucleotide.8
*Corresponding author. Fax:+1-617-353-3535; e-mail: tullius@bu.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00650-9

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B. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3093–3096
Scheme 1. Chemical/enzymatic synthesis of specifically deuterated nucleotides.
Scheme 2. Synthesis of [1-2H]-, [4-2H]-, and [5-2H2]-d-riboses.
with aqueous TFA to obtain deuterated d-ribose that
was suitable for enzymatic attachment to a purine base.
containing only trace quantities of the desired N-9-b
nucleoside. The necessity of carrying out several addi-
tional reactions to change the isopropyl protecting
group to acetyl groups on the sugar ring rendered this
approach unappealing.
The chemical synthesis of nucleosides has been trouble-
some because of the difficulty of obtaining regio- and
stereospecificity in the coupling of base to sugar. A
typical approach involves condensation of protected
and activated base and sugar.9ꢁ12 In a typical coupling
reaction, d-ribose is protected as 1,2,3,5-tetra-O-acetyl-,
acetyl-2,3,5-tri-O-benzoyl-b-d-ribofuranoside, or other
similar species. In our initial trials, coupling of an
anomeric mixture of isopropyl-protected d-ribose with
a silylated purine base resulted in complicated mixtures
We decided instead to use the procedure developed by
Tolbert and Williamson7 to phosphorylate and couple
deuterated ribose to a base. This one-pot method uses
an enzyme cocktail13 (ribokinase, PRPP synthetase,
myokinase, pyruvate kinase, enolase, 3-phosphoglycerate
mutase, and either adenine phosphoribosyltransferase, or
xanthine-guanine phosphoribosyltransferase plus guanylate

B. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3093–3096
3095
kinase) to convert unprotected d-ribose into a purine
nucleoside triphosphate under mild conditions with
good yield. Analytic HPLC using a Vydac 302IC4.6
HPLC column (anion exchange, 10 mm, 4.6 mm idꢂ250
mm) was used to monitor the progress of the enzymatic
reaction and to identify products. Purification of the
deuterated ribonucleoside triphosphate from the enzy-
matic reaction was performed by chromatography on
Affi-gel 601 (BioRad) boronate-derivatized poly-
acrylamide according to a published method.14 NMR
and mass spectrometry were used to characterize the
deuterated NT0P’s.15 For example, proton NMR spectra
of [10-2H]-, [4 -2H]- or [50-2H2]-nucleotides showed a
complete absence of the resonance for the 10-hydrogen
(d 5.73 or 5.93), the 40-hydrogen (d 4.15 or 4.21), or the
50-hydrogens (d 4.06 or 4.05), respectively, demonstrating
excellent incorporation of deuterium.
We used ribonucleotide reductase8,16,17 to transform a
specifically deuterated ribonucleotide into a 20-deoxyribo-
nucleotide that is suitable for incorporation into DNA
via the polymerase chain reaction. Formation of the
dNTP occurred relatively quickly. After 1.5 h, only a
trace of the unreacted starting material could be detected.
The reaction was stopped after 3 h. An aliquot (5 mL)
was removed and analyzed using a YMC ODS-AQ
HPLC column with a mobile phase of 0.1 M phosphate
(pH 6.5), at a flow rate of 0.7 mL/min, and detection at
260 nm (retention times: ATP, 11.60 min; dATP, 25.63
min; GTP, 6.09 min; dGTP, 12.35 min). The products were
purified on DEAE-Sephadex A-25 to afford deuterated
deoxyribonucleotides in yields of 74–76%. Deuterium
content was determined by mass spectrometry.
Figure 1. Denaturing polyacrylamide gel electrophoresis of the pro-
ducts of in vitro transcription of the gene for the L-21 Sca ribozyme.
Lane 1, RNA molecular weight markers. Lane 2, transcription of the
ribozyme gene was performed with [10-2H]-ATP. Lane 3, transcription
of the ribozyme gene was performed with [50-2H2]-ATP. Lane 4, tran-
scription of the ribozyme gene was performed with commercially
available, all-protio ATP (control). The intense band in lanes 2–4
corresponds to the expected 390 nt L-21 Sca ribozyme.
quantities useful for RNA transcription or DNA
synthesis.
Our aim in synthesizing specifically deuterated ribo-
nucleotides was to incorporate them into large RNA
molecules. To demonstrate this application, we carried
out in vitro transcription of the g0ene for the ScaI 0Tetra-
hymena ribozyme using either [1 -2H]-ATP or [5 -2H2]-
ATP, along with the other three natural nucleoside tri-
phosphates. As shown in Figure 1, aꢃ390 nt product
was obtained with either deuterated NTP (lanes 2and
3). The yield of deuterated RNA from the transcription
reaction was similar to that obtained when the reaction
was run with commercially available, all-protio ATP
(lane 4). These results clearly demonstrate the utility of
specifically deuterated ribonucleotides in making iso-
topically labeled RNA molecules.
Acknowledgements
This research was supported by PHS grant R01
GM40894 (TDT). National Research Service Award
F32GM20475 to E.R.J. is gratefully acknowledged. We
thank Professor JoAnne Stubbe and Professor Jamie
Williamson for generously supplying materials. We
thank Professor C. A. Townsend, Dr. J. J. Hangeland,
and Dr. L. G. Scott for helpful discussions, and Dr. D.
Young for the deuterium content determination. Mass
spectral data were provided by the Boston University
School of Medicine Mass Spectrometry Resource, which
is supported by NIH/NCRR Grant Nos. P41-RR10888
and S10-RR10493 to Professor C. E. Costello.
In conclusion, we have synthesized purine nucleotides
with specific deuterium labels at C-10, C-40, and C-50 by
a simple and short route. With slight modifications this
procedure could be used to produce pyrimidine nucleo-
tides, and it is easily adaptable to the synthesis of
nucleotides with labels in other positions of the ribose
or base moiety. Limited chemical synthesis was required
to accomplish isotopic substitution at the various sugar
positions.18 Enzymatic reactions efficiently coupled iso-
topically labeled ribose to a purine base to yield the
desired NTP. An enzymatic transformation was used to
convert ribonucleotides to deoxyribonucleotides. The
method presented here allows for the synthesis of a
variety of specifically labeled deuterated nucleotides in
References and Notes
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B. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3093–3096
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J=5.6 Hz), 4.03–4.11 (m, 2H); MS m/z 507.0 (507.15 calcd for
C10H14DN5O13P3); deuterium content 98%. [50-2H2]-Adeno-
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(508.12calcd for C 10H13D2N5O13P3); deuterium content 94%.
[10-2H]-Guanosine-50-triphosphate: yield, 74%; 1H NMR
(400 MHz, D2O) d 7.94 (s, 1H), 4.58 (d, 1H, J=6.8 Hz), 4.39
(m, 1H), 4.16 (m, 1H), 3.98–4.10 (m, 2H); MS m/z 523.0
(523.15 calcd for C10H14DN5O14P3); deuterium content 94%.
[40-2H]-Guanosine-50-triphosphate: yield, 79%; 1H NMR
(400 MHz, D2O) d 7.96 (s, 1H), 5.73 (d, 1H, J=6.0 Hz), 4.58
(dꢂd, 1H, J=6.0, 4.8 Hz), 4.37 (d, 1H, J=4.8 Hz), 3.99–4.10
(m, 2H); MS m/z 523.2 (523.15 calcd for C10H14DN5O14P3);
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13. 3-Phosphoglycerate mutase was purchased from Boehrin-
ger Mannheim. Enolase, myokinase, pyruvate kinase, and
guanylate kinase were purchased from Sigma. We obtained as
the generous gifts of Jamie Williamson bacterial expression
strains for ribokinase, PRPP synthetase, adenine phospho-
ribosyltransferase, and xanthine-guanine phosphoribosyl-
transferase. We expressed and purified these enzymes using the
procedures described in ref 7.
14. Batey, R. T.; Inada, M.; Kujawinski, E.; Puglisi, J. D.;
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15. [10-2H]-Adenosine-50-triphosphate: yield, 78%; 1H NMR
(400 MHz, D2O) d 8.35 (s, 1H), 8.08 (s, 1H), 4.58 (d, 1H,
J=5.2 Hz), 4.39 (m, 1H), 4.21 (m, 1H), 4.01–4.12 (m, 2H); MS
m/z 507.2(507.15 calcd for C 10H14DN5O13P3); deuterium
content 94%. [40-2H]-Adenosine-50-triphosphate: yield, 81%;
1H NMR (400 MHz, D2O) d 8.35 (s, 1H), 8.08 (s, 1H), 5.93 (d,
1
yield, 72%; H NMR (400 MHz, D2O) d 7.94 (s, 1H), 5.73 (d,
1H, J=5.6 Hz), 4.58 (m, 1H), 4.36 (m, 1H), 4.15 (m 1H); MS
m/z 524.0 (524.12 calcd for C10H13D2N5O14P3); deuterium
content 99%.
16. We obtained the ribonucleotide reductase expression
plasmid pSquire as a generous gift from JoAnne Stubbe. We
expressed and purified the enzyme as described in ref 17.
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18. To obviate the need to use chemical synthesis to produce
specifically deuterated riboses, the following deuterated riboses
may be obtained from Omicron Biochemicals (South Bend IN;
http://www.omicronbio.com): d-[1-2H]ribose, d-[2-2H]ribose,
d-[3-2H]ribose, d-[4-2H]ribose, and d-[5,50-2H]ribose.