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• ARTICLES •
January 2010 Vol.53 No.1: 1–8
doi: 10.1007/s11426-010-0012-4
Synthesis and crystal structure study of
2′-Se-adenosine-derivatized DNA
SHENG Jia, SALON Jozef, GAN JianHua & HUANG Zhen*
Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA
Received September 19, 2009; accepted October 9, 2009
*Corresponding author (email: Huang@gsu.edu)
The selenium derivatization of nucleic acids is a novel and promising strategy for 3D structure determination of nucleic acids.
Selenium can serve as an excellent anomalous scattering center to solve the phase problem, which is one of the two major bottlenecks in macromolecule X-ray crystallography. The other major bottleneck is crystallization. It has been demonstrated that
the incorporated selenium functionality at the 2′-positions of the nucleosides and nucleotides is stable and does not cause significant structure perturbation. Furthermore, it was observed that the 2′-Se-derivatization could facilitate crystallization of oligonucleotides with fast crystal growth and high diffraction quality. Herein, we describe a convenient synthesis of the
2′-Se-adenosine phosphoramidite, and report the first synthesis and X-ray crystal structure determination of the DNA containing the 2′-Se-A derivatization. The 3D structure of 2′-Se-A-DNA decamer [5′-GTACGCGT(2′-Se-A)C-3′]2 was determined at
1.75 Å resolution, the 2′-Se-functionality points to the minor groove, and the Se-modified and native structures are virtually
identical. Moreover, we have observed that the 2′-Se-A modification can greatly facilitate the crystal growth with high diffraction quality. In conjunction with the crystallization facilitation by the 2′-Se-U and 2′-Se-T, this novel observation on the
2′-Se-A functionality suggests that the 2′-Se moiety is sole responsible for the crystallization facilitation and the identity of nucleobases does not influence the crystal growth significantly.
selenium derivatization, nucleic acid, adenosine, X-ray crystallography, structuredetermination
1
Introduction
X-ray crystallography is one of the most powerful approaches for 3D structure determination of macromolecules,
including nucleic acids, proteins, and their complexes [1–4].
The 3D structure study at the atomic level provides novel
and detailed insights into the structure-function relationships, regulations, and molecular interactions of many biological processes. Besides the difficulty of crystallization,
however, heavy-atom derivatization of nucleic acids for
phase determination has largely slowed down determination
of new folds and structures, including nucleic acids, and
their complexes with drug-like small molecules and/or proteins [5, 6]. The conventional approaches, such as the heavyatom soaking and co-crystallization [7, 8], have proved to
© Science China Press and Springer-Verlag Berlin Heidelberg 2010
be much more difficult for nucleic acids than for proteins,
probably due to the lack of specific binding sites for metal
ions.
The halogen derivatization (especially Br), another conventional strategy, has been used by derivatizing DNAs and
RNAs with 5-bromo-deoxyuridine (thymidine analog) [9]
and 5-bromo-uridine [10] for multi- and single-wavelength
anomalous dispersion (MAD and SAD) phasing. The Br
derivatization was attempted as a perfect derivatization, in
combination with MAD or SAD phasing, for solving nucleic acid structures. However, the bromine derivatization is
primarily limited to the 5-position of uracil or cytosine due
to its chemical instability. In addition, the bromine functionality was found to be light-sensitive, and long-time exposure to X-ray or even ultraviolet (UV) sources may cause
decomposition [9–11]. More seriously, the Br derivatization
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2
SHENG Jia, et al.
Sci China Chem
can cause significant structure perturbation due to the
change of local hydration pattern in the major groove [12].
Therefore, it’s important and urgent to develop novel and
better alternative methodologies for derivatization and
phase determination [12–15].
The Se-methionine derivatization method [16–19] has
revolutionized the protein X-ray crystallography (Figure
1(a)). It is estimated that over two-thirds of novel protein
structures have been determined by the Se-methionine
MAD or SAD phasing [19]. Inspired by the Se-methionine
approach, our research group has pioneered and developed
the chemical and enzymatic incorporation of selenium functionalities into nucleic acids (see reviews [14, 15]) through
atom-specific replacement of oxygen with selenium (Figure
1(b)). This has opened up a novel research area, which has
attracted many attentions and research activities in chemical
synthesis, biochemistry and structural biology [12, 20–30].
So far, selenium has been introduced into several different
positions of DNAs and RNAs, including the 5′ [20], 2′ [12,
21, 26, 31, 32], and 4′ [28,33] positions of the ribose, the
phosphate backbone [34–37], and the nucleobases [27, 29,
30, 38].
Among them, the selenium modification at the 2′ position
(e.g., 2′-Se-Me) is the most stable and synthetically accessible. The 2′-Se-uridine phosphoramidite building block is
also commercially available [14]. Although the 2′-Seadenosine building block has been synthesized previously
[39], the synthesis is not practical for large-scale synthesis.
Furthermore, any structures and parameters of DNAs containing the 2′-Se-A derivatization haven’t been reported yet.
To address these critical issues, we describe here an efficient synthesis of the 2′-Se-adenosine phosphoramidite, and
report the first synthesis, crystallization study, and X-ray
crystal structure determination of the DNA containing the
2′-Se-A derivatization.
2
2.1
Experimental
General experimental section.
1
H NMR spectra were recorded on a Varian 400 MHz spec-
Figure 1 (a) Structure of native Met and Se-Met. (b) Atom specific selenium replacement of oxygen in nucleic acid.
January (2010) Vol.53 No.1
trometer. The chemical shifts are reported relative to TMS.
Analytical thin-layer chromatography (TLC) was performed
on silica 60F-254 plates. Flash column chromatography was
carried out on silica gel 60 (70–230 mesh). All reactions
were carried out under an argon atmosphere. The starting
material 9-[-D-arabinofuranosyl]adenine·H2O (Vidarabine)
was obtained from Berry & Associates, USA. Chemical
reagents and solvents were purchased from Aldrich and Alfa
Aesar, and were used without further purification with the
exception of these reagents: triethylamine (Et3N) and
N,N-diisopropylethylamine (DIPEA) were distilled from
KOH, and dimethoxytrityl chloride (DMT-Cl) was crystallized from hexanes. Only 1H NMR was performed for these
previously reported compounds [32].
2.2 Synthesis of the 2′-(Se-Me)-adenosine phosphoramidite
N6-acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)
( -D-arabinofuranosyl)-adenine (2)
Vidarabine 1 (2.85 g, 10 mmol) was co-evaporated three
times with dry pyridine (25 mL each) and then resuspended
in dry DMF (50 mL) and pyridine (50 mL) under an argon
atmosphere. To this suspension, 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (3.5 g, 11 mmol, 1.1 equiv) was added
dropwise. The mixture was stirred at room temperature for
2 h. Then, trimethylsilyl chloride (2.5 ml, 20 mmol, 2 equiv)
was added, and the reaction was stirred for another 2 h. After that, the reaction mixture was cooled in an ice bath, and
acetyl chloride (0.78 mL; 11 mmol, 1.1 equiv) was added
over 5 min. The resulting yellow solution was stirred at
room temperature for 1 h. The reaction mixture was then
poured into 150 mL of 5% aq. NaHCO3 and extracted with
dichloromethane (2 × 100 mL). The combined organic layers were washed with water, dried over MgSO4, and evaporated and co-evaporated three times with toluene. The crude
product was purified by flash column chromatography on
SiO2 (CH2Cl2/CH3OH, 100/0–99/1 v/v). Yield: 5.0 g of colorless foam (81% over three steps). This intermediate
6-acetyl-3′,5′-O-(1,1,3,3-tetraisopropyl-disiloxane-1,3-diyl)2′-O-(trimethylsilyl)-adenosine) was analyzed by 1H NMR for
purity monitoring, and was used for the next reaction. A
mixture of p-toluenesulfonic acid monohydrate (1.70 g, 8.80
mmol), dry dioxane (40 mL) and freshly dried molecular
sieves (4 g) was stirred at room temperature for 2.5 h. A
solution of the above intermediate (5.0 g, 8 mmol) in dioxane (20 mL) was added, and stirring was continued for 1 h.
The reaction mixture was quenched by the addition of neat
triethylamine (11.5 mL), evaporated, and co-evaporated
with dichloromethane. The crude product was purified by
flash column chromatography on SiO2 (CH2Cl2/CH3OH,
99.5/0.5–97/3 v/v). Yield: 3.30 g of 2 as colorless foam
(75%). 1H NMR (400 MHz, CDCl3):  1.05 (m, 28H, 2 ×
[(CH3)2CH]2Si), 2.58 (s, 3H, COCH3), 3.85 [d, J = 6.4 Hz,
SHENG Jia, et al.
Sci China Chem
1H, HO-C(2’)], 3.91 [m, J = 3.2, 8.0 Hz, 1H, H-C(4′)], 4.11
[m, 1H, H1-C(5′), 1H, H2-C(5′)], 4.64 [t, J = 8.0 Hz, 1H,
H-C(3′)], 4.73 [m, 1H, H-C(2′)], 6.30 [d, J = 6.4 Hz, 1H,
H-C(1′)], 8.32 [s, 1H, H-C(8)], 8.60 [s, 1H, H-C(2)], 8.83 (s,
1H, H-N6) ppm.
6
N -acetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)
-2′-methylseleno-2′-deoxyadenosine (3)
To a solution of compound 2 (3.30 g, 6 mmol) in CH2Cl2
(100 mL), 4-dimethylaminopyridine (2.20 g, 18 mmol, 3
equiv) was added at 0°C. The mixture was treated with
trifluoromethanesulfonyl chloride (0.96 ml, 9 mmol, 1.5
equiv) and stirred at 0°C for 10 min. The red reaction
mixture was diluted with CH2Cl2 (100 mL), washed with
5% aqueous NaHCO3, dried over MgSO4 and evaporated.
The crude product was used for the next step without further
purification. Sodium borohydride (0.57 g, 15 mmol, 2.5
equiv) was placed in a sealed 250 mL round-bottom flask,
dried on a high vacuum for 5 min to deplete oxygen, kept
under argon and suspended in dry THF (50 mL). Dimethyldiselenide (1.1 mL, 12 mmol, 2 equiv) was slowly injected
into this suspension, followed by dropwise addition of anhydrous ethanol (5 mL). The solution was stirred at room
temperature for 1 h, and the resulted almost colorless solution was injected into a solution of crude sulfonyl intermediate (prepared in situ) in dry THF (50 mL). The reaction
mixture was stirred at room temperature for 20 min. Then,
aqueous 0.2 M triethylammonium-acetate buffer (100 mL,
pH 7.4) was added, and the solution was reduced to half the
volume by evaporation. Dichloromethane (100 mL) was added, and the organic layer was separated. The water layer was
extracted again with CH2Cl2 (100 mL). The combined organic layer was dried over MgSO4 (s), and the solvent was
evaporated. The crude product was purified by flash column
chromatography on SiO2 (CH2Cl2/CH3OH, 100/0– 98/2 v/v).
Yield: colorless foam (3.0 g, 80% over two steps). 1H NMR
(400 MHz, CD2Cl2):  1.07 (m, 28H, 2 × [(CH3)2CH]2Si), 2.05 (s, 3H, SeCH3), 2.65 (s, 3H,COCH3), 4.15 [m,
1H, H-C(4′), 1H, H1-C(5′), 1H, H2-C(5′)], 4.23 [m, J = 6.8
Hz, 1H, H-C(2′)], 4.95 [t, J = 6.8 Hz, 1H, H-C(3′)], 6.37 [d,
J = 3.6 Hz, 1H, H-C(1′)], 8.28 [s, 1H, H-C(8)], 8.65 [s, 1H,
H-C(2)], 8.68 (s, br, 1H, H-N6) ppm.
N6-acetyl-2′-methylseleno-2′-deoxyadenosine (4)
Compound 3 (3.0 g, 4.8 mmol) was dissolved in a mixture of
1 M tetrabutylammonium fluoride/0.5 M acetic acid in THF
(15 mL). The solution was stirred at room temperature for
30 min. The solvent was evaporated and the residue dried
under high vacuum. The crude product was purified by flash
column chromatography on SiO2 (CH2Cl2/CH3OH, 99/1–97/3
v/v). Yield: 1.67 g of 4 as colorless foam (91%). 1H NMR
(400 MHz, DMSO-d6):  1.59 (s, 3H, SeCH3), 2.27 (s, 3H,
COCH3), 3.59 [m, 1H, H1-C(5′)], 3.68 [m, 1H, H2-C(5′)],
4.02 [m, 1H, H-C(4′)], 4.20 [dd, J = 5.2, 8.4 Hz, 1H,
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3
H-C(2′)], 4.39 [m, 1H, H-C(3′)], 5.13 [t, J = 5.6 Hz, 1H,
HO-C(5′)], 5.90 [d, J = 5.2 Hz, 1H, HO-C(3′)], 6.38 [d, J =
8.4 Hz, H-C(1′)], 8.68 [s, 1H, H-C(2)], 8.76 [s, 1H, H-C(8)],
10.73 (s, 1H, H-N6).
N6-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deo
xyadenosine (5)
Compound 4 (2.5 g, 6.5 mmol) was co-evaporated with dry
pyridine and then dissolved in pyridine (20 mL). Freshly
crystallized dimethoxytrityl chloride (2.85 g, 8.5 mmol, 1.3
equiv) was added in two portions over a period of 30 min.
The reaction mixture was stirred at room temperature for
1 h. The reaction was quenched by the addition of methanol
(0.5 mL). The solvents were removed under vacuum and the
residue was dissolved in dichloromethane, washed with
saturated NaHCO3 solution, dried over MgSO4 (s), and
evaporated. The crude product was purified by column
chromatography on SiO2 (CH2Cl2/ MeOH, 100/0–98/2 v/v).
Yield: 3.60 g of 5 as colorless foam (80%). 1H NMR (400
MHz, CD2Cl2):  1.95 (s, 3H, SeCH3), 2.65 (s, 3H, COCH3),
2.80 [s, br, 1H, HO-C(3′)], 3.48 [m, 1H, H1-C(5′), 1H,
H2-C(5′)], 3.81 (s, 6H, 2 × OCH3), 4.34 [m, 1H, H-C(4′)],
4.44 [m, 1H, H-C(2′)], 4.52 [m, 1H, H-C(3′)], 6.25 [d, J =
8.8 Hz, 1H, H-C(1′)], 6.84, 7.24–7.48 (m, 13H, trityl-H),
8.13 [s, 1H, H-C(8)], 8.54 [s, 1H, H-C(2)], 8.56 (s, br, 1H,
H-N6) ppm.
N6-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-methylseleno-2′-deoxyad-enosine-3′-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite (6)
A mixture of 5 (3.60 g, 5.2 mmol), N,N-diisopropyleth-ylamine (5.40 mL, 31.2 mmol, 6 eq.) in dry dichloromethane
(40 mL) was stirred under argon for 10 min. To the solution,
2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.90 g,
7.8 mmol, 1.5 eq.) was slowly added and the solution was
continued to stir at room temperature for 2 h. The reaction
mixture was concentrated in a vacuum to one-third of its
volume. The crude solution was then loaded onto a silica gel
column (pre-equilibrated with 2% Et3N in CH2Cl2) and
eluted (CH2Cl2/MeOH/Et3N, 96.9/0.1/3 v/v/v). The pooled
fractions containing pure phosphoramidite 6 were concentrated to dryness, dissolved in a minimum volume of dichloromethane and precipitated in hexanes (1.5 L). Yield:
3.35 g of 6 as colorless foam (72%). 1H NMR (400 MHz,
CDCl3, as a mixture of two diastereoisomers):  1.09–1.29
(m, 24H, 2 × [(CH3)2CH]2N), 1.62 (s, 3H, SeCH3), 1.70 (s,
3H, SeCH3), 2.36 (m, 2H, CH2CN), 2.67 (m, 8H,
COCH3,CH2CN), 3.40 [m, 2H, H1-C(5′)], 3.48–3.69 (m, 4H,
[(CH3)2CH]2N, 2H, H2-C(5′), 2H, POCH2), 3.79, 3.80 (2s,
12H, 2 × OCH3), 3.93–3.98, 4.14–4.27 (2m, 2H, POCH2),
4.45 [m, 4H, H-C(2′), H-C(4′)], 4.67, 4.73 [2m, 2H, H-C(3′)],
6.33 [m, 2H, H-C(1′)], 6.80, 7.22–7.48 (m, 26H, trityl-H),
8.14 [s, 1H, H-C(8)], 8.18 [s, 1H, H-C(8)], 8.55, 8.56 [2s,
2H, H-C(2)], 8.75 (s, br, 2H, H-N6) ppm.
4
2.3
SHENG Jia, et al.
Sci China Chem
Synthesis of the 2′-Se-A DNA decamer
The sequence of our target oligonucleotide for demonstration [5′-GTACGCGT(2′-Se-A)C-3′] was selected from the
PDB (protein data bank) [40] and chemically synthesized in
a 1.0 mol scale using an ABI3400 DNA/RNA Synthesizer.
The regular DNA phosphoramidite reagents were used in
this work (Glen Research).
The 2′-Se-modified dA-phosphoramidite was incorporated into oligonucleotides, with an additional reduction
step, using the standard protocol for solid-phase synthesis:
(i) coupling [phosphoramidites in dry acetonitrile (0.1 M)
activated by 0.3 M benzylthiotetrazole in dry acetonitrile],
(ii) capping (Ac2O/2,6-lutidine/THF, and 16% 1-methylimidazole/THF), (iii) oxidation (0.02 M I2/THF/Py/H2O), (iv)
reduction [DTT treatment (1 mL, 0.1 M DTT in EtOH/ H2O
= 2/3, for 2 min) after each capping-oxidation step], and (v)
detritylation (3% CCl3COOH in CH2Cl2). Solid-phase synthesis was performed on control pore glass (CPG-500) immobilized with the appropriate nucleoside (Glen Research).
The oligonucleotide was made in the DMTr-on mode. After
synthesis, the Se-DNA was cleaved from the solid support
and fully deprotected by conc. NH4OH at 55°C overnight.
After ammonia solution evaporation and HPLC purification,
the 5′-DMTr group was removed by treatment with the
aqueous solution of trichloroacetic acid (3% as the final
concentration) for 3 min, followed by hexane extraction.
The solution was then neutralized to pH 7.0 with a freshly
prepared 2 M TEAAC buffer and purified again with HPLC
for desalting. Alternatively, the Se-DNA was desalted on a
C18 SepPak cartridge (Waters/Millipore), washed with H 2O,
and eluted with H2O/CH3CN (6:4).
2.4
HPLC analysis and purification
The DNA oligonucleotide was analyzed and purified by
reverse-phase high performance liquid chromatography
(RP-HPLC) in both DMTr-on and -off forms. The purification was carried out using a 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 60% acetonitrile in buffer A. Similarly, the analysis was performed on a XB-C18 column
column (Welchrom, 4.6 × 250 mm) at a flow of 1.0 mL/min
using 10 mM TEAA (pH 7.6) as buffer A and 10 mM TEAA
in 50% acetonitrile (pH 7.6) as buffer B. The DMTr- on
oligonucleotide was purified by eluting with up to 100%
buffer B in 20 min in a linear gradient, starting from 10%
buffer B. The analysis for both the DMTr-on and DMTr-off
oligonucleotides was carried out with up to 70% of buffer B
in a linear gradient in 10 min, starting from 5% buffer B.
The collected fractions from preparative HPLC were combined and lyophilized to dryness.
2.5
Crystallization
The purified DNA oligonucleotide (1 mM) was heated to
January (2010) Vol.53 No.1
70°C for 2 min, and cooled down 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 using the hanging drop method by vapor diffusion.
2.6
Data collection
30% glycerol, PEG400 or the perfluoropolyether was used
as a cryoprotectant during the crystal mounting, and data
collection was taken under the liquid nitrogen stream at 99
K. The Se-DNA crystal data were collected at beam line
X12C in NSLS of Brookhaven National Laboratory. A
number of crystals were screened to find the one with the
strongest anomalous scattering signal at the K-edge absorption of selenium. The distance of the detector to the crystals
was set to 150 mm. The wavelengths of 0.9795 Å 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 using HKL2000 and DENZO/
SCALEPACK [41].
2.7
Structure determination and refinement
The structure of Se-DNA was solved by molecular replacement with CNS [42]. The refinement protocol includes
simulated annealing, positional refinement, restrained
B-factor refinement, and bulk solvent correction. The stereo-chemical topology and geometrical restrain parameters
of DNA/RNA [43] have been applied. The topologies and
parameters for modified adenosine (XUA) were constructed
and applied. After several cycles of refinement, a number of
highly ordered waters were added. Final, the occupancies of
selenium were adjusted. Cross-validation [44] with a 5%–
10% test set was monitored during the refinement. The Aweighted maps [45] of the (2m|Fo|-D|Fc|) and the difference
(m|Fo|-D|Fc|) density maps were computed and used
throughout the model building.
3
3.1
Results and discussion
Synthesis of 2′-Se-adenosine phosphoramidite
To synthesize the desired 2′-methylseleno-modified adenosine phosphoramidite (6), our synthesis was launched with
the well-established protection of the 3′- and 5′-hydroxyl
groups of commercially available Vidarabine 1 by a tetraisopropyldisiloxanyl group (TIPDS), followed by the trimethylsilyl protection of the 2′-hydroxyl group and the acetyl
protection of the adenosine 6-amino group. This intermediate was purified by column chromatography or crystallization, and then treated with 4-toluenesulfonic acid for the
selective removal of the 2′-trimethylsilyl group (Scheme 1).
T he formed N 6 -acet ylated arab ino nucle o sid e 2
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January (2010) Vol.53 No.1
5
tide was as efficient as our previous work [21, 26, 31]. We
synthesized a self-complementary DNA decamer [5′GTACGCGT(2′-Se-A)C-3’] [40] as a demonstration for the
synthesis, crystallization and crystal structure determination.
The analytical HPLC profile and MALDI-TOF mass spectrum of the purified DNA sample are showed in Figure 2.
Similar to the 2′-Se-U and 2′-Se-T work [12, 26], we performed the crystallization screening and studied the crystal
growth of this Se-modified oligonucleotide. We found that
this Se-DNA generated high-quality crystals in the Hampton Nuclei Acid Mini Screen kit. More excitingly, the crystals showed up overnight in 21 conditions out of all the 24
conditions of the kit. In addition, the crystals showed up in
all the 24 buffer conditions within two days. Since it was
demonstrated in our previous work that the corresponding
native DNA did not form crystals in any buffers of the kit
(24 buffers) over several weeks [12], we conclude here that
this 2′-Se-A modification greatly facilitates the crystal
growth of the DNA. The crystallization facilitation by the
2′-Se-U and 2′-Se-T has been observed previously [12, 26].
This novel observation of the 2′-Se-A crystallization facilitation suggests that the 2′-Se moiety is chiefly responsible
Scheme 1 Synthesis of 2′-(Se-Me)-adenosine phosphoramidite building
block. Reaction conditions: (i) 1.1 equiv TIPDSiCl2, DMF/Py (1/1), room
temperature, 2 h; (ii) 2 equiv TMS-Cl, room temperature, 2 h; (iii) 1.1
equiv Ac-Cl, room temperature, 1 h; (iv) 1.1 equiv PTSA.H2O, dioxane,
room temperature, 30 min, 61% over four steps; (v) 1.5 equiv CF3SO2Cl, 3
equiv. DMAP, CH2Cl2, 0 °C, 10 min; (vi) 2 equiv CH3SeSeCH3, 2.5 equiv
NaBH4, THF/EtOH, 20 min, room temperature, 80% over two steps; (vii) 1
M TBAF/ 0.5 M AcOH/ THF, room temperature, 1 h, 91%; (viii) 1.3 equiv
DMT-Cl, Py, room temperature, 1 h, 80%; (ix) 1.5 equiv 2-cyanoethylN,N-diisopropylchlorophosphoramidite, 6 equiv DIPEA, CH2Cl2, room
temperature, 2 h, 72%. (TIPDSiCl2: 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TMS-Cl: trimethylsilyl chloride; Ac-Cl: acetyl chloride;
PTSA.H2O: 4-toluenesulfonic acid monohydrate; CF3SO2Cl: trifluoromethane-sulfonyl chloride; DMAP: 4-(dimethylamino)pyridine; CH3SeSeCH3:
dimethyl diselenide; TBAF: tetrabutyl-ammonium fluoride; DMT-Cl:
dimethoxytrityl chloride: DIPEA: N,N-diisopropylethyl amine).
was triflated at the 2′-position to provide a good leaving
group for the following substitution (S N2) with sodium methylselenide, producing 2′-metylseleno compound 3 in high
yield. Deprotection of the TIPDS group went uneventfully
using tetrabutylammonium fluoride (TBAF) and acetic acid.
Compound 4 was allowed to react with dimethoxytrityl
chloride in order to protect the 5′-hydroxyl group. The final
step, transformation of 5 into the corresponding phosphoramidite 6, was accomplished by reaction with 2- cyanoethyl N,N-diisopropylchlorophosphoramidite [26, 31]. Our
simplified route leads to synthesis of phosphoramidite 6 in a
satisfactory overall yield with fewer synthetic and purification steps.
3.2
Crystallization facilitation by Se-derivatization
The incorporation of this 2′-Se-adenosien into oligonucleo-
Figure 2 The RP-HPLC (a) and MS (b) analyses of the 2’-SeA-modified DNA [GTACGCGT(2′-Se-A)C]. HPLC analysis conditions:
Welchrom XB-C18 column (4.6 × 250 mm) with a gradient of 5 to 60%
buffer B in 10 min, a flow rate: 1 mL/min, 25°C; A: 10 mM TEAAc (pH
7.1); B: 60% acetonitrile in 10 mM TEAAc (pH 7.1). MALDI-TOF (m/z):
the Se-DNA (molecular formular: C98H125N38P9O58Se), found [M+H]+:
3121.1 (calcd 3121.5); found [M + Na]+: 3143.1 (calcd. 3143.5).
6
SHENG Jia, et al.
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for the crystallization facilitation and the identity of nucleobases does not significantly influence the crystal growth.
January (2010) Vol.53 No.1
Table 1
Data collection and refinement statistics of the 2′-Se-A DNA
GTACGCGT(2’-Se-A)CAC (3IFF)
structure (PDB ID)
data collection
3.3
Data collection and structure analysis
The best data set for the Se-DNA structure determination
was collected from a crystal grown in buffer No. 23 of the
kit (10% v/v MPD, 40 mM Na-cacodylate, pH 7.0, 12 mM
spermine tetra-HCl, 40 mM lithium chloride, 80 mM strontium chloride). The crystal image is shown in Figure 3(a).
The data collection and structure refinement statistics are
summarized in Table 1. The Se-DNA structure resolution is
1.75 Å. As shown in Figure 3(b), this A-form Se-DNA
structure (in red, pdb ID: 3IFF) is virtually identical to the
native structure (in cyan, pdb ID: 395D). Both of them have
the same hexagonal space group. The overall r.m.s.d. of the
Se-DNA over the native is low (0.288), and the deviation is
mainly contributed by the first and last nucleotides. The
electron density of the 2′-Se-A:T base pair is shown in Figure 3(c), and the 2′-methylseleno functionality locates in
minor groove and doesn’t cause significant base-pair perturbation (Figure 3(d)). Furthermore, this structure provides
important information on the bond lengths of the C2′-Se and
Se-Me (1.98 and 1.96 Å, respectively), and on the bond
angles of the C1′-C2′-Se, C3′-C2′-Se and C2′-Se-Me (114o,
110o and 100o, respectively).
space group
cell dimensions: a,b,c (Å),
, ,  (°C)
resolution range (Å) (last shell)
P61
38.75, 38.75, 76.63,
90, 90, 120
30.0–1.65 (1.71–1.65)
unique reflections
7686 (681)
Completeness (%)
96.9 (86.4)
Rmerge (%)
5.0 (40.8)
I/(I)
41.5 (2.0)
redundancy
7.3 (2.9)
refinement
resolution range (Å)
30.0–1.75
Rwork, (%)
20.6
Rfree, (%)
27.8
number of reflections
6208
number of atoms
–
nucleic acid (double)
408
heavy atoms and ions
2 Se
water
59
R.m.s. deviations
–
bond length (Å)
0.011
bond angle
1.964
Rmerge = |I-‹I›|/I
Figure 3 Crystal picture and global and local structures of the 2′-Se-A DNA [5’-GTACGCGT(2’-Se-A)C-3’]2. (a) A typical crystal image. (b) The
Se-DNA structure (3IFF, in red) is superimposed over the native one (395D, in cyan), with a r.m.s.d. 0.288 Å, and the two yellow balls represent the selenium atoms. (c) The experimental electron density of Se-A: T base pair (= 1.0). (d) Se-A: T pair (in green) is superimposed over the native one (in cyan).
SHENG Jia, et al.
4
Sci China Chem
Conclusions
January (2010) Vol.53 No.1
11
In summary, we have developed a convenient synthesis of
the 2′-Se-adenosine phosphoramidite, and report the first
synthesis, crystallization study and X-ray crystal structure
determination of the DNA containing the 2′-Se-A derivatization at 1.75 Å resolution. This 2′-Se-moiety doesn’t cause
significant structure perturbation, the Se-modified and native A-form structures are virtually identical, and the
2′-Se-functionality points to the minor groove. Moreover,
we have observed that the 2′-Se-A modification can largely
facilitate the crystal growth with high diffraction quality.
Consistent with the crystallization facilitation by the
2′-Se-U and 2′-Se-T functionalities, this novel observation
of the 2′-Se-A-assisted crystallization suggests that the
crystal growth is not significantly influenced by the nucleobases, and the crystallization facilitation is mainly attributed to the 2′-Se functionality. This 2′-Se-modificantion
most likely locks the sugar pucker into the A-form DNA
conformation, a north conformation (or 2′-exo and 3′-endo
conformation). We have demonstrated that the 2′-Se-strategy can significantly facilitate the derivatization, phase determination, and crystallization of oligonucleotides. This
novel approach also has great potentials in 3D structure and
function studies of nucleic acid-protein complexes.
12
This work was financially supported by the US National Science Foundation (NSF MCB-0824837), and by the Georgia Cancer Coalition (GCC)
Distinguished Cancer Clinicians and Scientists.
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