Internal derivatization of oligonucleotides with selenium for x-ray crystallography using MAD

Article abstract of DOI:10.1021/ja0171097

We have developed a route for the synthesis of 2′-selenium uridine analogues and oligonucleotides containing selenium labels, and have demonstrated for the first time a new strategy to covalently derivatize nucleotides with selenium for phase and structur

Full text of DOI:10.1021/ja0171097

Published on Web 12/12/2001
Internal Derivatization of Oligonucleotides with Selenium for X-ray
Crystallography Using MAD
Quan Du,† Nicolas Carrasco,† Marianna Teplova,‡ Christopher J. Wilds,‡ Martin Egli,‡ and
Zhen Huang*,†
Department of Chemistry, Brooklyn College, and Program of Biochemistry and Chemistry, The Graduate School,
The City UniVersity of New York, 2900 Bedford AVenue, Brooklyn, New York 11210, and Department of Biological
Sciences, Vanderbilt UniVersity, NashVille, Tennessee 37235-1634
Received September 18, 2001
Determination of the three-dimensional structures of DNA
oligonucleotides, DNA-drug complexes, ribozymes, and viral
RNAs with high resolution is invaluable for gaining insights into
their functions and mechanisms.1-4 Several approaches, including
heavy-atom soaking, cocrystallization, and halogen derivatization,
have been used to label DNA and RNA for nucleic acid X-ray
crystallography.5 Heavy atom soaking and cocrystallization often
prove not very successful for nucleic acid X-ray crystallography,
and halogen derivatization is usually limited to short nucleotides.
Figure 1.
Scheme 1 a
In protein X-ray crystallography, selenium is used to replace sulfur
to mimic methionine,6 and this selenomethionine derivatization
method is widely used in phase and structure determination of
proteins using multiwavelengh anomalous dispersion (MAD).7
Indirect derivatization of RNAs using selenomethionine-labeled
RNA-binding protein U1A for phase and structure determination
has been successfully demonstrated, although RNA-binding protein
U1A has to be prepared and appropriate positions for inserting the
U1A-binding site have to be identified by constructing numerous
ribozyme constructs and screening their complexes with the
protein.4,8 Therefore, direct labeling of nucleotides with selenium
will largely simplify the derivatization effort and will facilitate
nucleic acid X-ray crystallography.
Oxygen atoms in nucleotides are the best choices for selenium
selective substitution to mimic natural nucleotides. Recently, we
have reported replacement of the 5′-oxygen of nucleosides with
selenium and synthesis of oligonucleotides containing selenium at
5′-termini.9 Demonstration of the stability of the selenium func-
tionality and its compatibility with the solid-phase phosphoramidite
chemistry prompted us to investigate the possibility of introducing
selenium to other positions, especially internal positions. Here we
describe the synthesis of oligonucleotides containing selenium at
the 2′-R-position of uridine 1 (Figure 1), and reveal crystallization
and structural studies of the selenium-containing oligodeoxyribo-
nucleotides.
a a. MsCl/THF/TEA, 95% yield; b. toluene/ tetrahexylammonium
hydrogen sulfate/ Na2CO3 (satd), 96% yield; c. (Bu)4N+ F-, 95% yield; d.
NaHSe, then CH3I, or NaSeCH3, 96%; e. PCl(OCH2CH2CN)N(iPr)2, 92%
yield.
required to protect the resulting selenol from oxidation. When
sodium methylselenide was used as the nucleophile to open the
tricyclic ring of 5, selenium-nucleoside 6 was obtained in 96%
yield with methyl protection, which prevents oxidation of the
selenium functionality. The selenide nucleophilic reactions were
conducted in THF solution, which avoided the ring-opening at the
2-position, resulting in substitution at the base.12 Compound 6 was
analyzed by MS, 77Se NMR, 2D-NMR, and NOE experiments to
confirm the stereochemistry and the structure. Nucleoside 6 was
finally converted to selenium-labeled phosphoramidite 7 in 92%
yield by reaction with 2-cyanoethyl N,N-diisopropyl-chlorophos-
phoramidite.
Using the phosphoramidite 7, DNA and RNA analogues contain-
ing selenium at the 2′-positions [DNA-octamer, 5′-GUSeGTACAC;13
DNA-decamer, 5′-GCGTAUSeACGC-3′;14 RNA-hexamer, and 5′-
r(CGUSeAC)dG35] were synthesized following standard solid-phase
synthesis. The potential for scale-up was demonstrated by 10 µmol
syntheses. As expected, the protected selenide functionality was
After mesylation of partially protected uridine 2 at the 2′-position
(Scheme 1), the mesyl group was displaced by the uracil exo-2-
oxygen in basic conditions.10 A two-phase reaction system (toluene
and aqueous Na2CO3), catalyzed by a phase-transfer catalyst, was
developed to facilitate the nucleophilic substitution; anhydro-uridine
4 was formed in 96% yield. Since our experiments indicated that
the bulky 3′-TBDMS group blocked selenide nucleophiles attacking
at 2′ position from the R-face, this group was removed by the
fluoride treatment. We also found that if NaHSe generated by
11
reduction of selenium metal with NaBH4 was used as the
nucleophile to attack 5 at the 2′-position, an additional step was
† The City University of New York.
‡ Vanderbilt University.
9
24 VOL. 124, NO. 1, 2002 J. AM. CHEM. SOC.
10.1021/ja0171097 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
minor groove and the C3′-C2′-Se-Me torsion angles adopt an
antiperiplanar conformation. Details of the structure determination
and refinement results will be reported elsewhere.
In conclusion, we have developed a route for the synthesis of
2′-selenium uridine analogues and oligonucleotides containing
selenium labels, and have demonstrated for the first time a new
strategy to covalently derivatize nucleotides with selenium for phase
and structure determination in X-ray crystallography. The 2′-R-
postion selenium derivatization retains the native C3′-endo con-
formation of A-Form DNA and RNA molecules. As the solid-phase
synthesis allows preparation of Se-RNA and Se-DNA in large
scales, unlike the phosphoroselenonate-mediated autoligation of
DNA strands,16 this approach is suitable for RNA and A-Form DNA
derivatization for X-ray crystallography. Selenium labels can also
be incorporated into a large RNA molecule via ligation of a
transcribed fragment and a synthetic fragment containing selenium
labels. This derivatization method may serve as an alternative
approach in phase and structure determination of RNA-protein
and DNA-protein complexes by derivatizing RNA and DNA
instead of proteins.
Figure 2. Electrospray MS of the octamer, 5′-GUSeGTACAC, molecular
formula C78H99N30O46P7Se, MW ) 2489.63 (including all isotopes).
Measured (expected) m/e: [M - 2H+]2- ) 1243.2 (1243.8); [M - 3H+]3-
) 828.5 (828.8); [M - 4H+]4- ) 621.2 (621.4); [M - 5H+]5- ) 496.8
(496.9).
Acknowledgment. We thank Dr. Xiangpeng Kong at NYU for
collecting diffraction data at Synchrotron Light Source at Brookhaven,
Dr. Hsin Wang at the Staten Island College for assisting in high-
field NMR data collection, and Dr. Soll and Dr. Edeninger at Hunter
College for assisting in MS data collection and DNA synthesis.
We are also grateful to Dr. Zdzislaw Wawrzak for help with data
collection. This work was supported by PSC-CUNY Research
Awards (69674-00-29 and 62392-00-31) and New Research
Dimension Fund (to Z.H.), and NIH (GM-55237 to M.E.).
1
Supporting Information Available: HRMS, H, 13C, 77Se NMR
data and 2D-NMR spectra of the nucleoside analogues, mass spectra
of the oligonucleotides, HPLC analysis, and crystallization (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
Figure 3. X-ray fluorescence spectrum of the decamer crystal. The
theoretical value for the Se K edge is 12.6578 keV (0.9795 Å).
References
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oxidation. The Se-oligonucleotides 1 with methyl protection were
purified by HPLC, and the selenium functionality was confirmed
by electrospray mass spectrometry. The MS spectrum of the
octamer, shown as an example, is displayed in Figure 2, where a
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were observed.
Crystallization conditions were screened, and diffraction quality
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technique. These X-ray structures confirmed the presence of the
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