Synthesis of a 2′-Se-thymidine phosphoramidite and its incorporation into oligonucleotides for crystal structure study

Article abstract of DOI:10.1021/ol062937w

(Chemical Equation Presented) To investigate nucleic acids with selenium derivatization for crystallography, we report the first synthesis of 2′-methylseleno-thymidine phosphoramidite and its incorporation into DNAs and RNAs by solid-phase synthesis with over 99% coupling yield. The d(GT _SeGTACAC)₂ crystal structure was also determined at 1.40 A resolution using Se phasing, revealing that this Se derivatization did not cause significant structure perturbation, consistent with our UV melting study. In addition, we observed that the Se modification largely facilitated the crystallization.

Full text of DOI:10.1021/ol062937w

ORGANIC
LETTERS
2007
Vol. 9, No. 5
749-752
Synthesis of a 2′-Se-thymidine
Phosphoramidite and Its Incorporation
into Oligonucleotides for Crystal
Structure Study
Jia Sheng, Jiansheng Jiang, Jozef Salon, and Zhen Huang*
Department of Chemistry, Georgia State UniVersity, Atlanta, Georgia 30303
huang@gsu.edu
Received December 4, 2006
ABSTRACT
To investigate nucleic acids with selenium derivatization for crystallography, we report the first synthesis of 2
′
-methylseleno-thymidine
phosphoramidite and its incorporation into DNAs and RNAs by solid-phase synthesis with over 99% coupling yield. The d(GTSeGTACAC)2
crystal structure was also determined at 1.40 Å resolution using Se phasing, revealing that this Se derivatization did not cause significant
structure perturbation, consistent with our UV melting study. In addition, we observed that the Se modification largely facilitated the crystallization.
Nucleic acids play key roles in genetic information storage,
expression, and translation and have attracted intense research
attention for a long time.1-4 X-ray crystallography has been
widely used to study the structure and function of nucleic
acid molecules.5,6 To facilitate crystal structure determination
of nucleic acids, especially using multiwavelength anomalous
dispersion (MAD) or single-wavelength anomalous disper-
sion (SAD) phasing,7 Huang, Egli, and co-workers were the
first to develop the selenium derivatization strategy.8-13 So
far, the 2′-Se derivatization has been introduced to U and C
by us and to A and G by other groups.9-15 Using the Se
derivatization and phasing, research laboratories have already
reported several crystal structures of DNA and RNA
molecules,10,14-16 including the homo-DNA structure, the
phasing of which otherwise proved extremely challenging.16
Natural tRNAs and rRNAs are heavily modified, and over
100 different modifications have been observed.17 Ribo-
(7) (a) Yang, W.; Hendrickson, W. A.; Crouch, R. J.; Satow, Y. Science
1990, 249, 1398-1405. (b) Hendrickson, W. A.; Ogata, C. M. Methods
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(8) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucleo-
tides Nucleic Acids 2001, 20, 1723.
(1) Nougayre`de, J.-P.; Homburg, S.; Taieb, F.; Boury, M.; Brzuszkiewicz,
E.; Gottschalk, G.; Buchrieser, C.; Hacker, J.; Dobrindt, U.; Oswald, E.
Science 2006, 313, 848.
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Nature 2004, 430, 45.
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E.; Ebright, Y. W.; Berman, H. M.; Ebright, R. H. Science 2002, 297, 1562.
(4) Watson, J. D.; Crick, G. H. C. Nature 1953, 171, 737.
(5) Lima, L. M. T. R.; Cordeiro, Y.; Tinoco, L. W.; Marques, A. F.;
Oliveira, C. L. P.; Sampath, S.; Kodali, R.; Choi, G.; Foguel, D.; Torriani,
I.; Caughey, B.; Silva, J. L. Biochemistry 2006, 45, 9180.
(6) (a) Ferre-D’Amare, A. R.; Zhou, K.; Doudna, J. A. Nature 1998,
395, 567. (b) Egli, M. Curr. Opin. Chem. Biol. 2004, 8, 580. (c) Holbrook,
S. R.; Kim, S. H. Biopolymers 1997, 44, 3.
(9) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z.
J. Am. Chem. Soc. 2002, 124, 24.
(10) (a) Teplova, M.; Wilds, C. J.; Wawrzak, Z.; Tereshko, V.; Du, Q.;
Carrasco, N.; Huang, Z.; Egli, M. Biochimie 2002, 84, 849. (b) Wilds, C.
J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. J. Am. Chem. Soc. 2002,
124, 14910.
(11) (a) Buzin, Y.; Carrasco, N.; Huang, Z. Org. Lett. 2004, 6, 1099.
(b) Carrasco, N.; Huang, Z. J. Am. Chem. Soc. 2004, 126, 448. (c) Carrasco,
N.; Buzin, Y.; Tyson, E.; Halpert, E.; Huang, Z. Nucleic Acids Res. 2004,
32, 1638. (d) Salon, J.; Chen, G. X.; Portilla, Y.; Germann, M. W.; Huang,
Z. Org. Lett. 2005, 7, 5645. (e) Logan, G.; Igunbor, C.; Chen, G.-X.; Davis,
H.; Simon, A.; Salon, J.; Huang, Z. Synlett 2006, 10, 1554.
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10.1021/ol062937w CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/31/2007

thymidine (rT) is a common modification in mature tRNAs
and rRNAs. Though the ribothymidine is the invariable
modification at position 54 of tRNAs of nearly all bacteria
and enkarya,17b its function in the TΨC loop has not been
well understood. Because our previous study indicated that
the Se derivatization at the 2′-positions of the nucleotides
does not cause significant perturbation of RNAs,9-11 the Se
incorporation at the 2′-position of ribothymidine in tRNAs
and rRNAs will help to obtain insight into the important rT
modification. Furthermore, the Se incorporation at the same
2′-position of thymidine will help to study the structure and
function of DNA molecules. Therefore, there is an urgent
need to synthesize the 2′-Se-thymidine derivative, as an
analogue of both ribothymidine and thymidine for the RNA
and DNA studies. We report here the first synthesis of the
2′-methylseleno-thymidine derivative and the corresponding
phosphoramidite, its incorporation into DNAs and RNAs by
solid-phase synthesis, and the UV melting and X-ray crystal
structure studies of the Se-derivatized DNAs and RNAs.
The synthesis of the 2′-methylseleno-ribothymidine de-
rivative (4) started from the glycosylation of the protected
D-ribose (1) with thymine under the Vorburggen conditions.18
Intermediate 3 was synthesized by minor modifications of
the literature procedures.19 In brief, due to the 2′-neighboring
group effect, this glycosylation generated exclusively the
protected â-ribothymidine derivative, followed by the NaOMe
treatment to give fully deprotected ribothymidine (2) in 88%
yield over two steps. After the intramolecular cyclization of
2 in DMF, the 2,2′-anhydrothymidine was selectively
protected with the DMTr group at the 5′-position to give 3
in 87% yield in two steps (Scheme 1). In the Se functionality
incorporation step, we initially encountered problems of low
yield and long reaction time in the Se introduction into
anhydroribothymidine 3. We attempted to increase the
reaction yield by protecting the 3′-hydroxyl group and using
different reducing reagents, such as LiAlH4 and DIBAH, to
form the methyl selenide nucleophile via the reduction of
dimethyl diselenide. Unfortunately, we observed the nucleo-
base reduction and the elimination reaction. These problems
were later overcome by using a weaker reducing reagent
(NaBH4) at a higher temperature (50 °C) in EtOH, which
gave 82% yield in 3 h. Finally, the 2′-Se-ribothymidine (4)
was converted to the corresponding phosphoramidite (5) in
90% yield.11a,c
Similar to solid-phase oligonucleotide synthesis using the
thymidine phosphoramidite,9,11a,c this novel 2′-Se-ribothymi-
dine phosphoramidite was incorporated into several DNA
and RNA oligonucleotides under the standard conditions in
the presence of 5-BMP as the coupling reagent. Analogous
to our previous report, the 2′-Se functionality on rT was
stable under the mild I2 treatment (20 mM, 20 s).9,11c The
synthesized oligonucleotides were cleaved off the solid
support and fully deprotected with concentrated ammonia
at 55 °C overnight, followed by reversed-phase HPLC
purification twice: DMTr-on and DMTr-off purification. A
typical HPLC profile of the Se-DNAs with the DMTr groups
is shown in Figure 1A, which is almost identical to that of
the corresponding native DNA (data not shown). The HPLC
Scheme 1. Synthesis of 5′-DMTr-2′-MeSe-thymidine
Phosphoramidite (5) and Oligonucleotides Containing This Se
Derivatization (6)
Figure 1. Reversed-phase HPLC analysis and purification of the
Se-DNAs. (A) The HPLC analysis profile of the crude DMTr-on
Se-DNA (5′-DMTr-GTSeGTACAC-3′) after cleavage from the solid
support and deprotection. The HPLC analysis was performed on a
Zorbax SB-C18 column (4.6 × 250 mm) with a linear gradient
from buffer A (20 mM triethylammonium acetate, pH 7.1) to 70%
buffer B (50% acetonitrile, 20 mM triethylammonium acetate,
pH 7.1) in 20 min; the Se-DNA retention time was 20.6 min.
(B) The HPLC purification profile of the DMTr-off Se-DNA (5′-
GTSeGTACAC-3′). The sample was eluted on a Zorbax SB-C18
column (21 × 250 mm; flow rate of 6 mL/min) with the same
gradient and buffers; its retention time was 14.8 min.
750
Org. Lett., Vol. 9, No. 5, 2007

analysis of the native and modified DNA octamers indicated
that the Se-modified and nonmodified phosphoramidites had
the same incorporation efficiency, revealing a high coupling
yield for the Se-T phosphoramidite (99%) as the total
formation of the short abortive oligonucleotides was less than
5%. A representative HPLC purification profile of the DMTr-
off Se-DNAs is shown in Figure 1B. All of the purified Se-
oligonucleotides were also confirmed by MALDI-TOF MS
analysis. A typical MS spectrum of the Se-oligonucleotides
is shown in Figure 2; all MS data of Se-DNAs and Se-RNAs
are collected in Table 1.
Table 1. MALDI-TOF MS Data of Se-oligonucleotides
entry
measured
m/z
Se-oligonucleotides
Se-DNA-1
(5′-GTSeGTACAC-3′)
79H101N30O46P7Se: FW 2502.5
Se-DNA-2
(5′-ATGGTSeGCTC-3′)
(calcd)
a
b
c
[M + H]+:
2503 (2503.5)
C
[M + H]+:
2823 (2823.8)
C89H114N32O54P8Se: FW 2822.8
Se-DNA-3
[M + H]+:
(5′-GGATSeGGGCG-3′)
2938 (2937.9)
C
91H112N42O51P8Se: FW 2936.9
d
e
f
Se-DNA-4
[M + H]+:
(5′-GCGTSeATACGC-3′)
3121 (3121.4)
C98H125N38O58P9Se: FW 3120.4
Se-RNA-1
(5′-GGTSeAUUGCGGUACC-3′)
[M + H]+:
4555 (4554.7)
C135H169N52O97P13Se: FW 4553.7
Se-RNA-2
[M + H]+:
(5′-UGAGCTSeUCGGCUC-3′)
4186 (4185.5)
C
124H157N45O91P12Se: FW 4184.5
g
Se-RNA-3
[M + H]+:
(5′-CUGUGTSeUCGAUCCACAG-3′)
5455 (5455.2)
C162H204N60O118P16Se: FW 5454.2
were collected by a Cary 300 UV-visible spectrophotometer
with a temperature controller at a heating rate of 0.5 °C/
min. The data were analyzed in accordance with the
convention of Puglisi and Tinoco.20 The melting temperatures
and typical curves are presented in Table 2 and the
Figure 2. MALDI-TOF MS analysis of the DMTr-off Se-DNA
(ATGGTSeGCTC). Molecular formula: C89H114N32O54P8Se. FW:
2822.8. Found [M + H]+: 2823. Calcd [M + H]+: 2823.8.
To compare the thermostability of the Se-oligonucleotides
with their native ones, the UV melting experiment was
carried out. The samples (2 µM DNA or RNA duplexes or
hairpin loop), dissolved in the buffer of 50 mM NaCl, 10
mM NaH2PO4-Na2HPO4 (pH 6.5), 0.1 mM EDTA, and 10
mM MgCl2, were heated to 85 °C and allowed to cool to
room temperature slowly. The oligonucleotide samples were
then stored at 5 °C for 2 h before the UV melting
measurements were carried out. All of the denaturing curves
Table 2. UV Melting Temperatures of the 2′-Se-RNAs and
-DNAs
melting temp
entry
DNA/RNA pairs
(°C, native)
RNA
a
b
c
5′-GGTSeAUUGCGGUACC-3′
3′-CCA--UAACGCCAUGG-5′
5′-UGAGCTSeUCGGCUC-3′
3′-ACUCGA--AGCCGAG-5′
5′-CUGUGTSeUCGAUCCACAG-3′
(the tRNA TΨC hairpin loop)
53.5 (54.2)
47.2 (47.8)
51.7 (52.4)
(13) Brandt, G.; Carrasco, N.; Huang, Z. Biochemistry 2006, 45, 8972.
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DNA
5′-ATGGTSeGCTC-3′
3′-TACCA--CGAG-5′
5′-GGATSeGGGCG-3′
3′-CCTA--CCCGC-5′
d
e
42.9 (42.6)
40.1 (40.2)
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Huang, Z.; Benner, S. A. J. Org. Chem. 2002, 67, 3996.
(19) (a) Mansuri, M. M.; Starrett, J. E.; Wos, J. A.; Tortolani, D. R.;
Brodfuehrer, P. R.; Howell, H. G.; Martin, J. C. J. Org. Chem. 1989, 54,
4780. (b) Saito, Y.; Zevaco, T. A.; Agrofoglio, L. A. Tetrahedron 2002,
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Supporting Information, respectively. There were no sig-
nificant differences between the melting temperatures of the
2′-Se-oligonucleotide duplexes and the native ones. In
addition, the Se-modified and native tRNA TΨC loops have
almost the same melting temperatures. Therefore, our UV
thermostability study indicates that this selenium modification
(20) Puglisi, J. D.; Tinoco, I., Jr. Methods Enzymol. 1989, 180, 304.
Org. Lett., Vol. 9, No. 5, 2007
751

does not significantly alter the duplex and loop structures,
which is consistent with our previous UV melting results of
the 2′-Se-DNA and RNA oligonucleotides.11a,c As the UV
melting study cannot provide detailed information on the
structure change, NMR structural analysis will be needed
for further characterization.
In our crystallization study, we found that the Se-DNA
(GTSeGTACAC, self-complementary) crystallized in 2-3
weeks in the native buffer, where the native DNA (GTG-
TACAC) crystallized over 2 months.21,22 When the Nucleic
Acid Mini Screen kit (Hampton Research, with 24 diversified
crystallization buffers) was screened, to our pleasant surprise,
we found that the Se-DNA crystallized overnight in 20 out
of 24 buffers (buffers no. 5-24). In contrast, the native DNA
did not crystallize at all over many weeks in these Hampton
buffers. It was also reported15b that the two RNAs (12-mer
and 16-mer) containing the 2′-Se-guanosines crystallized,
respectively, in 17 and 33 buffers, whereas their correspond-
ing native RNAs crystallized in 15 and 24 buffers, individu-
ally. Though the crystallization conditions of these Se-RNAs
expanded slightly, it appears that these two Se-RNAs, which
crystallized mostly as thin needle crystals, behaved similarly
to many Se-Met-derivatized proteins that required the fine
tuning of their native crystallization conditions. By contrast,
the majority of these Se-DNA crystals grown under the new
buffers was larger than the native DNA crystals grown under
the native buffer conditions, and these Se-DNA crystals also
diffracted well. Our results reveal that the 2′-Se derivatization
of thymidine facilitated the crystal packing and growth and
appeared to help expand the crystallization conditions.
Figure 3. DNA crystal structures (Se in red). (A) Superimposed
structure comparison of the native DNA (GTGTACAC, 1DNS in
green) and the Se-derivatized DNA (GTSeGTACAC, 2HC7 in cyan).
(B) The structure model and the electron density map of 2′-Se-
ribothymidine in the structure.
coupling yield. Our thermal denaturing study of the Se-
modified DNAs and RNAs also reveals that this Se deriva-
tization did not significantly change the stability of the
structures of the DNA and RNA duplexes and the tRNA
TΨC loop. Furthermore, our X-ray crystal structure study
with a Se-modified DNA octamer (GTSeGTACAC) shows
that the Se-derivatized DNA structure (1.40 Å resolution) is
virtually identical to the native structure (2.0 Å resolution)
with the same space group. More importantly, we observed
that this Se modification dramatically expanded the crystal-
lization conditions of the Se-DNA and largely facilitated
crystal growth while retaining the high diffraction quality.
In addition to DNA investigations, this 2′-Se-thymidine can
also serve as a ribothymidine analogue for the structure and
function studies of tRNAs and rRNAs. As the Se derivati-
zation can facilitate the phase determination and crystalliza-
tion, it will significantly impact the structure and function
studies of nucleic acids and their protein complexes using
X-ray crystallography. In addition, we are in the process of
further investigating the mechanism of the crystal growth
facilitated by the Se derivatization.
Finally, the structure of the Se-DNA crystal, grown under
the buffer no. 7, was determined at 1.40 Å resolution via
the Se SAD phasing and refinement. We found that the
determined Se-DNA structure (Figure 3) is superimposable
to the native structure (2.0 Å resolution) with the same space
group,21 indicating that this Se derivatization does not alter
the native structure significantly. In addition, this higher
structure resolution allows observation of ordered 43 water
molecules and other structure features. The Se-derivatized
structure reveals that the 2′-Se-furanose displays the 3′-endo
sugar pucker, which is consistent with the sugar pucker of
A-form DNAs and RNAs, and the 2′-methylseleno group is
placed in the minor groove of the duplex. Data collection,
phasing, and refinement statistics of the determined Se-DNA
structure (2HC7) are listed in the Supporting Information.
Acknowledgment. We thank Dr. Anand Saxena at NSLS
beamline X12C for his help in the data collection, and thank
Dr. Abdalla Hassan at Georgia State University for his
comments and discussion. This work was partially supported
by the GSU Research Program, the NIH (GM069703), and
the NSF (MCB-0517092).
In conclusion, we have successfully synthesized the 2′-
Se-thymidine phosphoramidite (5) as an analogue of both
thymidine and ribothymidine phosphoramidites for the solid-
phase Se-oligonucleotide synthesis. Subsequently, we have
incorporated it into DNAs and RNAs with over 99%
Supporting Information Available: Experimental pro-
1
cedures, H and 13C NMR, UV melting curves, and HRMS
analytical data. This material is available free of charge via
the Internet at http://pubs.acs.org.
(21) Jain, S.; Zon, G.; Sundaralingam, M. Biochemistry 1989, 28, 2360.
(22) Thota, N.; Li, X. H.; Bingman, C.; Sudaralingam, M. Acta Cryst.
1993, D49, 282.
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