Stereospecific syntheses of 3'-deuterated pyrimidine nucleosides and their site-specific incorporation into DNA.

Article abstract of DOI:10.1021/ol034102g

[reaction: see text] 2'-Deoxy-3'-deutero pyrimidines have been synthesized in high yields and incorporated into deoxyoligonucleotides using standard phosphoramidite chemistry. A key synthetic step is a stereospecific reduction of 3'-keto nucleosides using sodium triacetoxyborodeuteride to give 3'-deuterated thymidine and 2'-deoxy uridine nucleosides. Conversion of the corresponding phorphoramidites 7a and 7b to 4-triazolo derivatives has, for the first time, enabled incorporation of 2'-deoxy-3'-deutero cytidine and 2'-deoxy-3'-deutero-5-methyl cytidine into oligonucleotides.

Full text of DOI:10.1021/ol034102g

ORGANIC
LETTERS
2003
Vol. 5, No. 6
917-919
Stereospecific Syntheses of
3′-Deuterated Pyrimidine Nucleosides
and Their Site-Specific Incorporation
into DNA
Panadda Chirakul and Snorri Th. Sigurdsson*
Department of Chemistry, UniVersity of Washington, Seattle, Washington 98195-1700
sigurdsson@chem.washington.edu
Received January 20, 2003
ABSTRACT
2′-Deoxy-3′-deutero pyrimidines have been synthesized in high yields and incorporated into deoxyoligonucleotides using standard
phosphoramidite chemistry. A key synthetic step is a stereospecific reduction of 3′-keto nucleosides using sodium triacetoxyborodeuteride
to give 3′-deuterated thymidine and 2′-deoxy uridine nucleosides. Conversion of the corresponding phorphoramidites 7a and 7b to 4-triazolo
derivatives has, for the first time, enabled incorporation of 2′-deoxy-3′-deutero cytidine and 2′-deoxy-3′-deutero-5-methyl cytidine into
oligonucleotides.
The importance of protein interactions with the phospho-
diester backbone of DNA is becoming increasingly apparent
as more high-resolution structures of protein/DNA complexes
reveal numerous direct protein contacts with the sugar-
phosphate backbone.1,2 It is likely that the structurally
redundant sugar-phosphate backbone plays a role in sequence-
specific recognition and that the local mobility of the
backbone is a component of the recognition mechanism. For
example, the results from molecular dynamics simulations
have suggested that the BI to BII DNA conformational change
can provide an indirect readout mechanism for DNA.3,4 We
are interested in applying solid-state deuterium NMR, whose
line shapes and relaxation times give information about the
dynamic state of individual torsion angles, to study the ꢀ
and ú torsion angles that are strongly correlated to the BI to
BII transition. However, this requires the sequence-specific
incorporation of 3′-deutero nucleosides into DNA, which has
not been extensively studied.5
The most obvious synthetic approach for deuteration in
the 3′-position of nucleosides is through reduction of the
corresponding 3′-keto nucleosides with a deuterated metal
hydride. However, this approach has two drawbacks. First,
the 3′-keto nucleosides are not very stable because of their
susceptibility to â-elimination of the nucleobase, especially
when cytosine is the base.6-8 Second, the 3′-ketone is
preferentially reduced from the R-face of the ketone, yielding
2′-deoxyxylonucleosides. For example, 3′-deutero-5′-O-tri-
tylthymidine was prepared in only 9% yield from the
corresponding ketone.7 Another recently reported synthetic
strategy is 3′-labeling of a protected 3-oxoribose, followed
(1) Klimasauskas, S.; Kumar, S.; Roberts, R. J.; Cheng, X. Cell 1994,
76, 357-369.
(5) Foldesi, A.; Trifonova, A.; Kundu, M. K.; Chattopadhyaya, J.
Nucleosides Nucleotides 2000, 19, 1615-1656.
(2) O’Gara, M.; Horton, J. R.; Roberts, R. J.; Cheng, X. Nat. Struct.
Biol. 1998, 5, 872-877.
(6) Crews, R. P.; Baker, D. C. Nucleosides Nucleotides 1983, 2, 275-
289.
(3) Wellenzohn, B.; Flader, W.; Winger, R. H.; Hallbrucker, A.; Mayer,
E.; Liedl, K. R. J. Am. Chem. Soc. 2001, 123, 5044-5049.
(4) Wellenzohn, B.; Flader, W.; Winger, R. H.; Hallbrucker, A.; Mayer,
E.; Liedl, K. R. Biochemistry 2002, 41, 4088-4095.
(7) Hansske, F.; Madej, D.; Robins, M. J. Tetrahedron 1984, 40, 125-
135.
(8) Robins, M. J.; Samano, V.; Johnson, M. D. J. Org. Chem. 1990, 55,
410-412.
10.1021/ol034102g CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/21/2003

by glycosylation to give 3′-deuteriothymidine in 19% overall
yield after 11 synthetic steps.9
Scheme 2. Synthesis of 3′-Deuterated Cytidine Derivative 6
We describe here the stereospecific synthesis of 2′-deoxy-
3′-deutero pyrimidine nucleosides in high yields and their
incorporation into DNA. This route is efficient and suitable
for large-scale preparation of pyrimidine nucleosides 1a and
1b (Scheme 1). The synthetic strategy utilizes the free 5′-
Scheme 1. Syntheses of 1a and 1b
from the â-face (Figure S1b) to yield a mixture of 1-(2′-
deoxy-3′-deutero-â-D-ribofuranosyl)thymine (1a) and 1-(2′-
deoxy-3′-deutero-â-D-xylofuranosyl)thymine (2a) (95:5 ratio
by 1H NMR), readily separated by column chromatography
or HPLC. Deuterium incorporation at the 3′-position of the
sugar ring was evident by the absence of the signal at 4.27
ppm from its 1H NMR spectrum. The 5′-hydroxyl group of
1a was subsequently protected as a dimethoxytrityl (DMT)
ether and the 3′-hydroxyl group phosphitylated to yield
phosphoramidite 7a, which was incorporated into DNA using
standard phosphoramidite chemistry.
The same strategy that was used to prepare 3′-deutero
thymidine was applied in the synthesis of 2′-deoxy-3′-deutero
cytidine, the synthesis of which has not been reported.
Previous attempts to prepare the keto derivative by oxidation
of 4-N-acetyl-2′-deoxy-5′-O-trityl cytidine with CrO3/pyri-
dine/Ac2O resulted in â-elimination and formation of 4,5-
dihydro-5-trityloxymethylfuran-4-one.7 In contrast, oxidation
of 4-N-benzoyl-2′-deoxy-5′-O-(dimethoxytrityl)cytidine14 with
PDC afforded the corresponding keto-derivative 5 in 75%
hydroxyl groups of 3′-keto thymidine and 2′-deoxy-3′-keto
uridine to direct reduction to the â-face of the 3′-ketone
through coordination with sodium triacetoxyborodeuteride.10,11
Selective oxidation of the 3′-hydroxyl group required the use
of a 5′-hydroxyl protecting group that could be removed after
oxidation without elimination of the nucleobase, because the
free 5′-hydroxyl group is required for directing the reducing
agent to the â-face of the ketone. Since the reductions are
performed under acidic conditions, we chose the acid-labile
dimethoxytrityl protecting group.
Oxidation of 5′-dimethoxytrityl thymidine was accom-
plished with pyridinium dichromate (PDC), resulting in 81%
yield of the corresponding 3′-keto thymidine (4a).12,13
Following oxidation, ketone 4a was deprotected and reduced
by treatment with sodium triacetoxyborodeuteride, formed
in situ from sodium borodeuteride and acetic acid, in a two-
step, one-pot procedure to give 1a in 85% yield. Figure S1
in Supporting Information illustrates the effect of sodium
triacetoxyborodeuteride on the change in stereoselectivity of
the reduction, when compared to sodium borodeuteride. The
sodium borodeuteride reduction preferentially reduced the
ketone from the R-face (Figure S1a),7 whereas sodium
triacetoxyborodeuteride delivered the hydride predominantly
1
yield, which was characterized by H NMR and HRMS.
However, the 3′-keto derivative of 2′-deoxy cytidine (5) was
much more unstable under the conditions of deprotection/
reduction than the corresponding thymidine derivative 4a.
This is presumably due to the ease of protonation of cytidine
under the acidic conditions, which accelerates â-elimination
of the nucleobase. As a result, the expected product 6 was
obtained in only 10% yield.
These results led us to investigate an alternative synthetic
route, utilizing the known conversion of a thymine base to
a methylcytosine through the corresponding triazole deriva-
tive (Scheme 3).15,16 2′-Deoxy-3′-keto uridine (4b) was
synthesized by the oxidation of 2′-deoxy-5′-O-dimethoxytri-
tyl uridine (3b) with PDC in 80% yield. Subsequent treatment
with triacetoxyborodeuteride resulted in a mixture of 1-(2′-
deoxy-3′-deutero-â-D-ribofuranosyl)uracil (1b) and 1-(2′-
deoxy-3′-deutero-â-D-xylofuranosyl)uracil (2b) (94:6 ratio
by 1H NMR). Compound 1b was isolated in 83% yield and
converted to the protected phosphoramidite 7b, from which
(9) Chen, T.; Greenberg, M. M. Tetrahedron Lett. 1998, 39, 1103-1106.
(10) Hoveyda, A. H.; Evans, D. A.; Fu, G. C. Chem. ReV. 1993, 93,
1307-1370.
(11) Robins, M. J.; Sarker, S.; Samano, V.; Wnuk, S. F. Tetrahedron
1997, 53, 447-456.
(12) Froehlich, M. L.; Swartling, D. J.; Lind, R. E.; Mott, A. W.;
Bergstrom, D. E. Nucleosides Nucleotides 1989, 8, 1529-1535.
(13) Svendsen, M. L.; Wengel, J.; Kirpekar, F.; Roepstorff, P. Tetrahe-
dron 1993, 49, 11341-11352.
(14) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982,
104, 1316-1319.
(15) Cowart, M.; Gibson, K. J.; Allen, D. J.; Benkovic, S. J. Biochemistry
1989, 28, 1975-1983.
(16) Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J. Org. Chem. 1992, 57, 3839-
3845.
918
Org. Lett., Vol. 5, No. 6, 2003

prepared from 2′-deoxy-3′-deutero thymidine (1a), to study
the effect of 2′-deoxycytidine methylation on the mobility
of the sugar by deuterium solid-state NMR (Scheme 3).
Scheme 3. Incorporation of 3′-Deuterated dC into DNA
In summary, we have shown that stereospecific reduction
of 3′-keto nucleosides is a short, high-yielding synthetic route
to 3′-deuterated thymidine and 2′-deoxy uridine nucleosides.
Conversion of the corresponding phorphoramidites 7a and
7b to 4-triazolo derivatives has, for the first time, enabled
incorporation of 2′-deoxy-3′-deutero cytidine and 2′-deoxy-
3′-deutero-5-methyl cytidine into oligonucleotides. Deuterium
solid-state NMR experiments containing these 3′-deuterated
nucleotides at specific sites in biologically relevant DNA
sequences are underway, and those results will be reported
in due course.
Acknowledgment. This work was supported by the
National Institutes of Health (GM 58914). The authors thank
Dr. G. P. Drobny for helpful discussions and Dr. M. Sadilek
for HRMS-FAB analyses.
the 2′-deoxy-3′-deutero-4-triazolo uridine phosphoramidite
8b was prepared in quantitative yield. This monomer was
incorporated into oligodeoxyribonucleotides using standard
automated DNA synthesis protocols. After deprotection and
purification of the oligomer, ESI-MS analysis verified the
incorporation of deuterium, and enzymatic digestion, fol-
lowed by analytical RP-HPLC, showed the return of the
nucleosides in their expected ratios (see Supporting Informa-
tion). 2′-Deoxy-3′-deutero-5-methyl cytidine was also in-
corporated into oligomers using the triazole derivative 8a,
Supporting Information Available: Experimental pro-
cedures and full characterization for compounds and 1H, 13
C
and 31P NMR spectra for new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL034102G
Org. Lett., Vol. 5, No. 6, 2003
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