Synthesis and crystal structure of 2'-Se-modified guanosine containing DNA

Article abstract of DOI:10.1021/jo902190c

(Chemical Equation Presented) Selenium modification of nucleic acids is of great importance in X-ray crystal structure determination and functional study of nucleic acids. Herein, we describe a convenient synthesis of a new building block, the 2′-SeMe-modified guanosine (G_Se) phosphoramidite, and report the first incorporation of the 2′-Se-G moiety into DNA. The X-ray crystal structure of the 2′-Se-modified octamer DNA (5′-GTG_SeTACAC-3′) was determined at a resolution of 1.20 A.We also found that the 2′-Se modification points to the minor groove and that the modified and native structures are virtually identical. Furthermore, we observed that the 2′-Se-G modification can significantly facilitate the crystal growth with respect to the corresponding native DNA.

Full text of DOI:10.1021/jo902190c

pubs.acs.org/joc
Synthesis and Crystal Structure of 20-Se-Modified Guanosine Containing
DNA
Jozef Salon, Jia Sheng, Jianhua Gan, and Zhen Huang*
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303
huang@gsu.edu
Received October 11, 2009
Selenium modification of nucleic acids is of great importance in X-ray crystal structure determination
and functional study of nucleic acids. Herein, we describe a convenient synthesis of a new building
block, the 20-SeMe-modified guanosine (GSe) phosphoramidite, and report the first incorporation
of0 the 20-Se-G moiety into DNA. The X-ray crystal structure of the 20-Se-modified octamer DNA
0
0
˚
(5 -GTGSeTACAC-3 ) was determined at a resolution of 1.20 A. We also found that the 2 -Se
modification points to the minor groove and that the modified and native structures are virtually
identical. Furthermore, we observed that the 20-Se-G modification can significantly facilitate the
crystal growth with respect to the corresponding native DNA.
Introduction
convenient method for the determination of novel nucleic
acid structures. However, the lack of multiple choices of
derivatization positions, light sensitivity,7 and potential
structure perturbations8 have limited its application in this
field. Determination of three-dimensional structures of
nucleic acids through indirect protein derivatization is ap-
parently labor intensive. Therefore, it is necessary to develop an
alternative method for nucleic acid structure determination.
Inspired by the Se-methionine derivatization of proteins,
which plays the important role in novel protein crystal
structure determination,9,10 the Se modification of nucleic
acids11,12 has recently been recognized as an important tool
in phasing X-ray diffraction data. We pioneered this research
X-ray crystallography plays a leading role in providing
structure information about nucleic acids with atomic reso-
lution.1 In this field, however, there are two long-standing
challenges: crystallization and the phase problem. Besides
molecular replacement, other strategies have been developed
to calculate the experimental phase of nucleic acid structures,
including heavy-atom soaking and cocrystallization in com-
bination with MIR or SIR (multiple- and single-isomor-
phous replacement), respectively.2-4 Halogen derivatization
and indirect protein derivatization have also been used in
conjunction with MAD or SAD (multi- and single-wavelength
anomalous dispersion), respectively.2-4 Halogen derivatization
strategy (especially with bromine)2,5,6 was thought to be a
(7) Ennifar, E.; Carpentier, P.; Ferrer, J. L.; Walter, P.; Dumas, P. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1262–1268.
(8) Jiang, J.; Sheng, J.; Carrasco, N.; Huang, Z. Nucleic Acids Res. 2007,
35, 477–485.
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1990, 249, 1398–1405. (b) Hendrickson, W. A.; Horton, J. R.; LeMaster,
D. M. EMBO J. 1990, 9, 1665–1672.
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W. A. Trends Biochem. Sci. 2000, 25, 637–643.
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Williams, J.; Huang, Z. Chem. Biodiversity 2008, 5, 396–407.
(12) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucleo-
tides Nucleic Acids 2001, 20, 1723–1734.
*To whom correspondence should be addressed. Phone: þ1-404-413-
5535. Fax: þ1-404-413-5505.
(1) Egli, M.; Pallan, P. S. Annu. Rev. Biophys. Biomol. Struct. 2007, 36,
281–305.
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2171–2176.
(3) Ferre-D’Amare, A. R.; Zhou, K.; Doudna, J. A. Nature 1998, 395,
567–574.
(4) Boggon, T. J.; Shapiro, L. Structure 2000, 8, 143–149.
(5) Tavale, S. S.; Sobell, H. M. J. Mol. Biol. 1970, 48, 109–123.
(6) Shah, K.; Wu, H.; Rana, T. M. Bioconjugate Chem. 1994, 5, 508–512.
DOI: 10.1021/jo902190c
r
Published on Web 01/04/2010
J. Org. Chem. 2010, 75, 637–641 637
2010 American Chemical Society

Salon et al.
JOCArticle
SCHEME 1. Synthesis of the 20-Selenomethyl-20-deoxyguanosine Phosphoramidite Building Block
area of selenium-derivatized nucleic acid (SeNA) for nucleic
acid crystallography. We and others have developed the
chemical and enzymatic syntheses of SeNAs.13-21 Among
several different Se modifications, whether on the sugar or
on the nucleobase,13-21 the 20-SeMe derivatization
(including the 20-SeMe-G in RNA)19 is quite stable and
preferred for its genuine ability to dramatically facilitate
crystal growth.8,22-24 To further explore the 20-Se-G deriva-
tization of nucleic acids, we report here a convenient synth-
esis of a new 20-Se-guanosine phosphoramidite for DNA and
RNA derivatization, describe the first incorporation of the
20-Se-guanosine moiety into DNA, and report the crystal-
lization and 3D crystal structure of 20-SeMe-G-derivatized
DNA.
Results and Discussion
In our approach, we first protected both the 30- and 50-
hydroxyl groups of commercially available 9-[β-D-arabino-
furanosyl]guanine using the tetraisopropyldisiloxanyl
(TIPDS) group. Then, we used a modified Beigelman proce-
dure to protect the guanosine amino group (N2) by the
isobutyryl group in excellent yield. The procedure utilizes
TMS transient protection of the 20-hydroxyl group and O6,
which is known to be prone to acylation and sulfonylation
reactions.25 Silylation of the O6 not only hinders O6 acylation,
but also drives N2 acylation, as it only requires 1.1 equiv of
isobutyryl chloride reagent.26 Upon the successful acylation, we
proceeded with a selective deprotection of the 20-O-TMS group
using 2 equiv of neat trifluoroacetic acid to furnish intermediate
2. The next step did not require protection of the O6 because we,
unlike others,19 found it to be nonreactive toward trifluoro-
methanesulfonyl chloride (CF3SO2Cl). In addition to a 61%
reaction yield, we were able to recover ca. 27% of the unreacted
starting material. Isolated derivative 3 was treated with in situ
generated sodium methylselenide, followed by deprotection of
the TIPDS group with tetrabutylammonium fluoride (TBAF)
to yield diol 5. Compound 5 was actually synthesized without
purification of 4 (66% yield in two steps). This key intermediate
was allowed to react with dimethoxytrityl chloride to produce
compound 6, which was subsequently converted into phos-
phoramidite 7 by reaction with 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite in the presence of dimethylethyl amine
(Scheme 1). Our novel approach supplies phosphoramidite 7
in a 21% overall yield in nine steps with only five steps of
chromatographic purification. We should mention that the
synthesis is scalable, allowing preparation of phosphoramidite
(13) (a) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang,
Z. J. Am. Chem. Soc. 2002, 124, 24–25. (b) Wilds, C. J.; Pattanayek, R.; Pan,
C.; Wawrzak, Z.; Egli, M. J. Am. Chem. Soc. 2002, 124, 14910–14916.
(14) Hobartner, C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.; Polonskaia,
A.; Serganov, A.; Micura, R. J. Am. Chem. Soc. 2005, 127, 12035–12045.
(15) Brandt, G.; Carrasco, N.; Huang, Z. Biochemistry 2006, 45, 8972–8977.
(16) Carrasco, N.; Buzin, Y.; Tyson, E.; Halpert, E.; Huang, Z. Nucleic
Acids Res. 2004, 32, 1638–1646.
(17) Salon, J.; Jiang, J.; Sheng, J.; Gerlits, O. O.; Huang, Z. Nucleic Acids
Res. 2008, 36, 7009–7018.
(18) Carrasco, N.; Caton-Williams, J.; Brandt, G.; Wang, S.; Huang, Z.
Angew. Chem., Int. Ed. Engl. 2006, 45, 94–97.
(19) (a) Moroder, H.; Kreutz, C.; Lang, K.; Serganov, A.; Micura, R.
€
J. Am. Chem. Soc. 2006, 128, 9909–9918. (b) Hobartner, C.; Micura, R.
J. Am. Chem. Soc. 2004, 126, 1141–1149. (c) Puffer, B; Moroder, H.; Aigner,
M; Micura, R. Nucleic Acids Res. 2008, 36, 970–983.
(20) Caton-Williams, J.; Huang, Z. Angew. Chem., Int. Ed. 2008, 47,
1723–1725.
(21) Watts, J. K.; Johnston, B. D.; Jayakanthan, K.; Wahba, A. S.; Pinto,
B. M.; Damha, M. J. J. Am. Chem. Soc. 2008, 130, 8578–8579.
(22) Salon, J.; Sheng, J.; Jiang, J.; Chen, G.; Caton-Williams, J.; Huang,
Z. J. Am. Chem. Soc. 2007, 129, 4862–4863.
(23) Hassan, A. E. A.; Sheng, J.; Jiang, J.; Zhang, W.; Huang, Z. Org.
Lett. 2009, 11, 2503–2506.
(24) (a) Sheng, J.; Jiang, J.; Salon, J.; Huang, Z. Org. Lett. 2007, 9, 749–
752. (b) Sheng, J.; Salon, J.; Gan, J.-H.; Huang, Z. Science China Chemistry
2010, 53, 78–85.
(25) Bridson, P. K.; Markiewicz, W. T.; Reese, C. B. J. Chem. Soc., Chem.
Commun. 1977, 791–792.
(26) Fan, Y.; Gaffney, B. L.; Jones, R. A. Org. Lett. 2004, 6, 2555–2557.
638 J. Org. Chem. Vol. 75, No. 3, 2010

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FIGURE 1. Crystal picture and structure of the Se-DNA (50-GTGSeTACAC-30)2: (A) typical crystal image and (B) Se-G:C base pair shown
with the electron density map contoured at the 1σ level.
A-form Se-DNA structure (3IFI, in red) is superimposed
over the native A-form DNA structure (1DNS, in cyan),
which has the same tetragonal space group P43212. The
overall rmsd of the Se-DNA over the native DNA is low
(0.39) and mainly contributed by the first and last bases, and
the main structure is considered the same as the native
structure. It is clear that the 20-SeMe derivatization points
into the minor groove and does not cause significant structure
perturbation with respect to the native structure. The detailed
data statistics for the structural analysis of this structure are
summarized in Table S1 (see the Supporting Information).
Probably due to higher molecular dynamics at the minor
groove region, the ordered water molecules are not observed
in both the native and Se-DNA structures. Consistent with
the previous 20-Se-dU- and T-DNA structures,8,24 we did not
find any interactions between the selenium atom (or the Se-
methyl group) and other atoms. Furthermore, we have
analyzed the duplex packing pattern, but no significant
intermolecular and/or molecular packing interactions were
found. Thus, the facilitated crystallization is likely due to the
intrinsic sugar pucker after the Se modification.
FIGURE 2. Crystal structure of the 20-Se-modified DNA (50-
GTGSeTACAC-30): (A) Superimposition of the 20-Se-modified
(red) and nonmodified (cyan) double-stranded DNAs with rms
0.39; the two red balls represent the selenium atoms. (B) Super-
imposition of the Se-G:C base pair (green) over the native G:C base
pair (cyan).
7 on a multigram scale, and is suitable not only for modification
of DNA but also RNA.
Conclusion
Similar to the 20-SeMe-T and 20-SeMe-U research,8,22-24
we performed a screening to study the crystallization beha-
vior of the modified oligonucleotide d(50-GTGSeTACAC-30)
as a model. We found that the 20-Se-G-DNA could generate
high-quality crystals in all the buffer conditions of the
Hampton kit (24 buffers) within 3 days (up to approximate
size 0.1 ꢀ 0.1 ꢀ 0.1 mm3). This size is sufficient for X-ray
diffraction data collection. In contrast, we found that the
native counterpart did not form crystals in any buffer of
the kit over several months.8 Interestingly, the crystal struc-
ture of the 20-Se-G-modified DNA has the highest resolution
of any other SeNAs. In addition, the 20-Se-T- and 20-Se-U-
modified DNAs with the same sequence did not crystallize in
all of the Hampton buffers,8,24 and their crystal structure
resolution was not as high as that of the 20-Se-G0-modified
In summary, we have developed a new and scalable
synthesis of 20-selenomethyl-20-deoxyguanosine phosphora-
midite 7, described the first incorporation of the 20-Se-G moiety
into DNA, and reported the X-ray crystal structure of the
0
˚
2 -Se-G modified DNA at a resolution of 1.20 A, which is the
highest structural resolution among the Se-derivatized nucleic
acids. It has also been found that this modification does not
cause any significant structure perturbation and that the
Se-modified and native structures are virtually identical.
Furthermore, we have observed that the 20-Se-G modification
speeds up crystal growth. Consistent with our previous work on
the 20-Se-U and 20-Se-T modifications, the 20-Se-G-assisted
crystallization success suggests that the crystal growth is mainly
reinforced by the 20-SeMe functionality itself instead of the
nucleobases. Our crystallization study, therefore, has potential
to aid in the structural and functional studies of nucleic acids as
well as nucleic acid-protein complexes.
˚
DNA (1.20 A). Therefore, we conclude that the 2 -SeMe-G
modification can greatly facilitate crystal growth of the
DNA (50-GTGSeTACAC-30) and that the 20-Se-G modifica-
tion may particularly be useful for crystallization facilitation
and preparation of crystals with high diffraction quality.
The data set of the best crystals, grown in buffer No. 12
(10% MPD, 40 mM Sodium Cacodylate pH 6.0, 12 mM
Spermine tetra-HCl, 80 mM KCl, and 20 mM BaCl2),
was chosen to determine the Se-DNA structure (Figure 1),
Experimental Section
N2-Isobutyryl-9-[30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-β-D-arabinofuranosyl]guanine (2). 9-[30,50-O-(1,1,3,3-Tetra-
isopropyldisiloxane-1,3-diyl)-β-D-arabinofuranosyl]guanine
(2.7 g, 9.5 mmol), prepared according to the literature,20 was
coevaporated with dry pyridine (50 mL). Dry dichloromethane
˚
which was resolved at 1.20 A. As shown in Figure 2, this
J. Org. Chem. Vol. 75, No. 3, 2010 639

Salon et al.
JOCArticle
(200 mL) and pyridine (50 mL) were added under argon, and the
mixture was cooled in an ice bath with stirring. Trimethylsilyl
chloride (7.2 mL, 57 mmol, 6 equiv) was added, and the flask was
removed from the ice bath and stirred for 2 h. The reaction
mixture was cooled again in an ice bath, and isobutyryl chloride
(1.1 mL, 10.5 mmol, 1.1 equiv) was added over 5 min. The
mixture was stirred at room temperature for 1.5 h. The solution
was then poured into 300 mL of 5% aq NaHCO3, and the
organic layer was isolated. The mixture was evaporated to a
thick oil followed by coevaporation with toluene (2 ꢀ 50 mL).
The fully protected intermediate was crystallized by addition of
diethyl ether (25 mL) and hexanes (25 mL). The crystalline
compound (5.6 g) was dissolved in dry CH2Cl2 (100 mL) by
means of ultrasonication. Into this solution, trifluoroacetic acid
(1.2 mL, 2 equiv) in dry dioxane (25 mL) was injected slowly.
The reaction mixture was stirred for 6 h. Neat triethylamine (2.3 mL)
was added and the mixture was filtered and concentrated. The
crude product was purified by flash column chromatography on
SiO2 (CH2Cl2/MeOH, 99.5/0.5-98/2 v/v). Yield: 4.03 g (71%
over four steps) of 2 as a colorless foam. 1H NMR (DMSO-d6):
δ 1.02-1.16 (m, 34H), 2.77 (m, 1H), 3.82 (m, 1H), 3.94 (dd, J =
2.8, 12.8 Hz, 1H), 4.04 (dd, J = 3.6, 12.8 Hz, 1H), 4.33 (t, J =
8.0 Hz, 1H), 4.50 (m, 1H), 5.85 (d, J = 6.4 Hz, 1H), 6.05 (d, J =
6.4 Hz, 1H), 7.91 (s, 1H), 11.75 (s, 1H), 12.08 (s, 1H) ppm.
N2-Isobutyryl-9-{20-O-[(trifluoromethyl)sulfonyl]-30,50-O-(1,
1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-D-arabinofuranosyl}-
guanine (3). A suspension of compound 2 (4.03 g, 6.8 mmol) in
CH2Cl2 (50 mL) was cooled to 4 °C in an ice water bath. DMAP
(2.50 g, 17 mmol, 2.5 equiv) was added, followed by dropwise
addition of trifluoromethanesulfonyl chloride (0.65 mL, 10.2 mmol,
1.5 equiv) over 2 min. The reaction mixture was then stirred at
4 °C for 10 min. It was diluted with dichloromethane, washed
with saturated sodium bicarbonate solution, dried over MgSO4,
and evaporated. The crude product was purified by flash column
chromatography on SiO2 (CH2Cl2/MeOH, 99.5/0.5-98/2 v/v).
The yield was 3.0 g (61%) of 3 as a colorless solid. Additionally,
11.70 (s, 1H), 12.11 (s, 1H), ppm. 13C NMR (DMSO-d6): δ 3.0,
19.3, 35.2, 46.8, 62.1, 73.3, 87.8, 88.8, 120.5, 138.2, 148.8, 149.5,
155.3, 180.6 ppm. ESI-TOF high-acc (m/z): calcd for
C15H21N5O5Se [M þ H]þ 432.0781, found 432.0777.
N2-Isobutyryl-30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-20-methylseleno-20-deoxyguanosine (4). 1H NMR (CDCl3):
δ 1.11 (m, 28H), 1.28, 1.30 (2 ꢀ s, 6H), 1.92 (s, 3H), 2.69 (m, 1H),
3.94 (dd, J = 4.4, 6.8 Hz, 1H), 4.05 (m, 2H), 4.16 (m, 1H), 4.74
(t, J = 6.8 Hz, 1H), 6.21 (d, J = 4.4 Hz, 1H), 7.93 (s, 1H), 8.57
(s, 1H), 12.11 (s, 1H), ppm. 13C NMR (CDCl3): δ 3.5, 12.6,
13.1, 13.2, 13.5, 16.9, 16.99, 17.0, 17.2, 17.3, 17.3, 17.4, 17.5,
19.0, 19.04, 34.7, 47.7, 61.8, 71.6, 84.5, 89.2, 121.6, 136.6, 147.7,
147.8, 155.6, 178.9 ppm. ESI-TOF high-acc (m/z): calcd for
C27H47N5O6SeSi2 [M þ H]þ 674.2303, found 674.2304; [Mþ
Na]þ 696.2122, found 696.2126.
N2-Isobutyryl-50-O-(4,40-dimethoxytrityl)-20-methylseleno-20-
deoxyguanosine (6). Compound 5 (1.20 g, 2.79 mmol) was
coevaporated with dry pyridine and then dissolved in pyridine
(10 mL). The solution was treated with dimethoxytrityl chloride
(1.23 g, 3.63 mmol, 1.3 equiv) in two portions over a period of
45 min. The reaction mixture was stirred for 1 h. The solvents
were removed under vacuum and the residue was dissolved in
dichloromethane, washed with 5% citric acid, water, and 5%
NaHCO3, and then dried over MgSO4 and evaporated. The
crude product was purified by column chromatography on SiO2
(CH2Cl2/MeOH/Et3N, 99/0/1-98/1/1 v/v/v). Yield: 1.71 g of
6 as a colorless foam (84%). 1H NMR (DMSO-d6): δ 1.13, 1.14
(2 ꢀ s, 6H), 1.68 (s, 3H), 2.76 (m, 1H), 3.10 (m, 1H), 3.38 (m, 1H),
3.74 (s, 6H), 4.07 (m, 1H), 4.20 (m, 1H), 4.25 (m, 1H), 5.89
(d, J = 4.0 Hz, 1H), 6.22 (d, J = 7.6 Hz, 1H), 6.84, 7.20-7.38
(m, 13H), 8.20 (s, 1H), 11.60 (s, 1H), 12.10 (s, 1H), ppm. 13C
NMR (CDCl3): δ 4.1, 18.5, 18.6, 36.0, 48.1, 55.2, 64.1, 72.7, 85.5,
86.3, 89.3, 113.1, 121.5, 127.0, 127.9, 128.1, 130.0, 135.7, 135.9,
138.3, 145.0, 147.9, 148.9, 155.7, 158.8, 179.6 ppm. ESI-TOF
high-acc (m/z): calcd for C36H39N5O7Se [M þ H]þ 734.2087,
found 734.2085; [M þ Na]þ 756.1907, found 756.1908.
N2-Isobutyryl-50-O-(4,40-dimethoxytrityl)-20-methylseleno-20-
deoxyguanosine 30-(2-Cyanoethyl)-N,N-diisopropylphosphorami-
dite (7). Compound 6 (1.50 g, 2.05 mmol) was dissolved in a
mixture of dimethylethyl amine (2.0 mL, 13.2 mmol, 6 equiv)
and dry dichloromethane (10 mL) under argon. After 10 min,
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.73 g,
3.1 mmol, 1.5 equiv) was slowly added and the solution was
stirred at room temperature for 2 h. The crude product was
purified by column chromatography on SiO2 (CH2Cl2/MeOH/
Et3N, 98/0/2-97.5/0.5/2 v/v/v). Yield: 1.68 g of 7 as a colorless
foam (88%). 1H NMR (CDCl3, two sets of peaks as a mixture of
two diastereoisomers): δ 0.56, 0.66, 0.78, 0.83 (d, 12H);
0.98-1.32 (m, 24H); 1.47 (m, 2H); 1.69, 1.74 (s, 6H); 2.16, 2.33
(2m, 2H); 2.66, 2.78 (t, m, 2H); 3.12 (m, 2H); 3.50-3.64 (m, 8H);
3.78, 3.79 (2s, 12H); 3.91-4.02, 4.11-4.24 (2 m, 2H); 4.27, 4.35
(m, 2H); 4.50 (m, 2H); 4.73, 4.78 (2 m, 2H); 6.00, 6.06 (2d, J =
9.6 Hz, 2H); 6.82, 7.26, 7.46, 7.58 (4m, 26H); 7.82, 7.83 (2s, 2H);
11.96 (s, 2H) ppm. 13C NMR (CDCl3): δ 3.5, 4.1, 18.3, 18.4,
18.4, 18.5, 20.4, 20.5, 24.4, 24.5, 24.6, 24.7, 35.9, 36.0, 43.2, 43.3,
47.0, 46.1, 55.3, 55.3, 57.5, 57.7, 63.3, 63.5, 74.7, 75.8, 84.9, 85.3,
86.1, 86.3, 91.1, 92.1, 113.3, 113.3, 122.7, 122.9, 127.2, 128.0,
128.10, 128.1, 129.97, 130.00, 130.04, 135.6, 135.8, 135.9, 136.2,
117.2, 117.6, 139.0, 144.9, 145.1, 147.0, 147.1, 148.3, 148.3,
155.4, 158.8, 178.3 ppm. 31P NMR (CDCl3): δ 149.3, 150.5 ppm.
ESI-TOF high-acc (m/z): calcd for C45H56N7O8PSe [M þ H]þ
934.3166, found 934.3170; [M þ Na]þ 956.2985, found 956.2983.
Synthesis of 20-SeMe-G Containing DNA. The sequence of
our target oligonucleotide (50-GTGSeTAC AC-30) was selected
from the PDB (protein data bank)27 and chemically synthesized
1
1.10 g (27%) of the starting material was isolated. H NMR
(DMSO-d6): δ 0.94-1.15 (m, 34H), 2.80 (m, 1H), 3.95 (d, J =
12.4 Hz, 1H), 4.03 (m, 1H), 4.21 (dd, J = 4.8, 12.4 Hz, 1H), 5.00
(t, J = 8.0 Hz, 1H), 6.10 (m, 1H), 6.38 (d, J = 6.4 Hz, 1H), 8.26
(s, 1H), 11.69 (s, 1H), 12.16 (s, 1H) ppm.
N2-Isobutyryl-20-methylseleno-20-deoxyguanosine (5). Sodium
borohydride (0.32 g, 2 equiv) was placed in a sealed 100 mL
round-bottomed flask, dried on a high vacuum for 5 min to
deplete oxygen, kept under argon, and suspended in dry THF
(25 mL). Dimethyl diselenide (0.77 mL, 2 equiv) was slowly
injected into this suspension, followed by the addition of
anhydrous ethanol (2.5 mL). The solution was stirred at room
temperature for 1 h. To this slightly yellow solution, compound
3 (3.0 g, 4.1 mmol) in dry THF (25 mL) was injected. The
reaction mixture was stirred at room temperature for 20 min.
Then, coldwater (50mL) was addedandthe solutionwas reduced
to half of its volume by evaporation. Dichloromethane (100 mL)
was added and the organic layer was separated. The water layer
was extracted twice with dichloromethane. The combined organic
layers were dried over MgSO4 and the solvent was evaporated to
dryness. A small portion of an oily substance was purified by
flash column chromatography on SiO2 (CH2Cl2/MeOH, 99/
0-99/1 v/v) to obtain compound 4 as a colorless foam. The oily
substance was dissolved in THF (20 mL) and treated with 1 M
TBAF in THF (6 mL). The solution was stirred at room
temperature for 5 min. The solvent was evaporated and the
product was isolated by flash column chromatography on SiO2
(CH2Cl2/MeOH, 99/1-95/5 v/v). The yield was 1.20 g (66%
over two steps) of 5 as a colorless solid. 1H NMR (DMSO-d6): δ
1.12, 1.14 (2 ꢀ s, 6H), 1.64 (s, 3H), 2.78 (m, 1H), 3.56 (m, 2H),
3.95 (m, 1H), 4.03 (m, 1H), 4.32 (m, 1H), 5.04 (t, J = 5.6 Hz, 1H),
5.84 (d, J = 4.8 Hz, 1H), 6.14 (d, J = 9.2 Hz, 1H), 8.31 (s, 1H),
(27) Jain, S.; Zon, G.; Sundaralingam, M. Biochemistry 1989, 28,
2360–2364.
640 J. Org. Chem. Vol. 75, No. 3, 2010

Salon et al.
JOCArticle
2 min, and then cooled slowly to room temperature. Both native
buffer and Nucleic Acid Mini Screen Kit (Hampton Research)
were applied to screen the crystallization conditions at different
temperatures (5, 10, and 20 °C), using the hanging drop method
by vapor diffusion.
Data Collection. The cryoprotectants 30% glycerol, PEG 400,
or perfluoropolyether were used during the crystal mounting,
and data collection was taken under a stream of liquid nitrogen
at 99 K. The 20-SeMe-dG-DNA crystal data were collected at
beamline X12B and X12C in the NSLS of the Brookhaven
National Laboratory. A number of crystals were scanned to find
the one with strong anomalous scattering at the K-edge absorption
of selenium. The distance of the detector to the crystals was set to
˚
150 mm. The wavelength 0.9795 A was chosen for selenium
SAD phasing. The crystals were exposed for 10 to 15 s per image
with one degree oscillation, and a total of 180 images were taken
for each data set. All the data were processed with HKL2000
and DENZO/ SCALEPACK.28
FIGURE 3. Representative HPLC analysis of the 20-Se-G-modi-
fied DNA (50-GTGSeTACAC-30). HPLC conditions: Welchrom
XB-C18 column (4.6 ꢀ 250 mm) with a gradient of 5-60% buffer
B in 10 min; flow rate 1 mL/min, 25 °C: (A) 10 mM TEAAc (pH 7.6);
(B) 60% acetonitrile in 10 mM TEAAc (pH 7.6). Inset: MALDI-
TOF (m/z): the Se-DNA (molecular formula: C79H101N30P7O46Se),
found [M þ H]þ 2503.4 (calcd. 2503.4).
Structure Determination and Refinement. The structure of
Se-DNA was solved by molecular replacement with both
CNS29 and Phaser.30 The refinement protocol includes simu-
lated annealing, positional refinement, restrained B-factor re-
finement, and bulk solvent correction. The stereochemical
topology and geometrical restraint parameters of DNA/
RNA31 have been applied. The topologies and parameters for
the modified guanosine with 20-SeMe (XUG) were constructed
and applied. After several cycles of refinement, a number of
highly ordered waters were added. Finally, the occupancies of
selenium were adjusted. Cross-validation32 with a 5-10% test set
was monitored during the refinement. The σA-weighted maps33
of the (2m|Fo| - D|Fc|) and the difference (m|Fo| - D|Fc|) density
maps were computed and used throughout the model building.
on a 1.0 μmol scale using a DNA Synthesizer. The 20-SeMe-
modified guanosine phosphoramidite 7 was incorporated into
oligonucleotides by using the standard protocol for solid-phase
synthesis with the additional step of treatmeant with DTT;14
coupling: phosphoramidites in dry acetonitrile (0.1 M) were
activated by 0.3 M benzylthiotetrazole in dry acetonitrile;
capping: (A) Ac2O/2,6-lutidine/THF, (B) 16% 1-methylimida-
zole/THF; oxidation: 0.02 M I2/THF/Py/H2O; detrytilation:
3% CCl3COOH in CH2Cl2; manual DTT treatment between
each coupling step (2 min): 0.1 M DTT (1 mL each time) in
EtOH/H2O (2/3). Solid-phase synthesis was performed on con-
trol pore glass (CPG-500) immobilized with the appropriate
nucleoside. The oligonucleotide was made in the DMTr-on
mode. After the synthesis, the Se-DNA oligonucleotide was
cleaved from the solid support and fully deprotected by concd
NH4OH at 55 °C.
Acknowledgment. This work was supported by the U.S.
National Science Foundation (NSF MCB-0824837) and the
Georgia Cancer Coalition (GCC) Distinguished Cancer
Clinicians and Scientists.
Note Added after ASAP Publication. This paper was pub-
lished on the Web on January 4, 2010, with an error in reference 24.
The corrected version was reposted on January 29, 2010.
HPLC Analysis and Purification. DNA oligonucleotides were
analyzed and purified by reverse-phase high-performance liquid
chromatography (RP-HPLC) in both DMTr-on and -off forms
(Figure 3). Purification was carried out with an XB-C18 column
(Welchrom, 21.2 ꢀ 250 mm) at a flow rate of 6 mL/min. Buffer A
consisted of 30 mM triethylammonium acetate (TEAA, pH 7.6),
while buffer B contained 50% acetonitrile in 30 mM TEAA
(pH 7.6). Analysis was performed on an XB-C18 column
(Welchrom, 4.6 ꢀ 250 mm) at a flow rate of 1.0 mL/min, using
buffers A and B. DMTr-on oligonucleotide was purified by
eluting with up to 100% B in 20 min in a linear gradient starting
at 5% B, while analysis for both the DMTr-on and -off
oligonucleotides was carried out with up to 70% of B in a linear
gradient in 10 min, starting at 5% B as well. The collected
fractions were combined, lyophilized, and desalted by a Sep-Pak
C18 cartridge.
Supporting Information Available: 1H NMR spectra of
compounds 2-7, 13C NMR spectra of compounds 4-7, 31P
NMR spectrum of compound 7, MALDI-TOF spectrum of the
20-Se-DNA (dGTACGSe CGTAC), and ESI-TOF high-acc
mass spectra of compounds 4-7. This material is available free
of charge via the Internet at http://pubs.acs.org.
(28) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307–326.
(29) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu,
N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystal-
logr., Sect. D: Biol. Crystallogr. 1998, 54, 905–921.
(30) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.;
Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658–674.
(31) Parkinson, G.; Vojtechovsky, J.; Clowney, L.; Brunger, A. T.;
Berman, H. M. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1996, 52, 57–64.
(32) Brunger, A. T. Nature 1992, 355, 472–475.
Crystallization. The purifiedDNAoligonucleotide(50-GTGSe-
TACAC-30) at a concentration of 1 mM was heated to 80 °C for
(33) Read, R. J. Acta Crystallogr. 1986, A42, 140–149.
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