Synthesis of selenium-derivatized nucleosides and oligonucleotides for X-ray crystallography

Article abstract of DOI:10.1081/NCN-100105907

We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach t

Full text of DOI:10.1081/NCN-100105907

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SYNTHESIS OF SELENIUM-DERIVATIZED
NUCLEOSIDES AND OLIGONUCLEOTIDES FOR X-RAY
CRYSTALLOGRAPHY
Nicolas Carrasco a , Dov Ginsburg a , Quan Du a & Zhen Huang b
a Department of Chemistry , Brooklyn College, and Program of Biochemistry and Chemistry,
The Graduate School of The City University of New York , 2900 Bedford Ave., Brooklyn, New
York, 11210, U.S.A.
b Department of Chemistry , Brooklyn College, and Program of Biochemistry and Chemistry,
The Graduate School of The City University of New York , 2900 Bedford Ave., Brooklyn, New
York, 11210, U.S.A.
Published online: 21 Aug 2006.
To cite this article: Nicolas Carrasco , Dov Ginsburg , Quan Du & Zhen Huang (2001) SYNTHESIS OF SELENIUM-DERIVATIZED
NUCLEOSIDES AND OLIGONUCLEOTIDES FOR X-RAY CRYSTALLOGRAPHY, Nucleosides, Nucleotides and Nucleic Acids, 20:9,
1723-1734, DOI: 10.1081/NCN-100105907
To link to this article: http://dx.doi.org/10.1081/NCN-100105907
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(9), 172371734 (2001)
SYNTHESIS OF SELENIUM-DERIVATIZED
NUCLEOSIDES AND OLIGONUCLEOTIDES
FOR X-RAY CRYSTALLOGRAPHY
Nicolas Carrasco, Dov Ginsburg, Quan Du, and Zhen Huang*
Department of Chemistry, Brooklyn College, and Program of
Biochemistry and Chemistry, The Graduate School of The City
University of NewYork, 2900 Bedford Ave., Brooklyn,
NewYork 11210
ABSTRACT
We report here the synthesis of nucleoside and oligonucleotide analogs
containing selenium, which serves as an anomalous scattering center to
enable MAD phase determination in nucleotide X-ray crystallography.
We have developed a phase transfer approach to introduce the selenium
functionality in A, C, G, T, and U nucleosides at 50-positions. In the
incorporation of the selenium functionality, the leaving groups (bromide,
mesyl, and tosyl) were readily displaced by sodium selenide, sodium
diselenide, and sodium methyl selenide with yields higher than 90%.
Selenium-derivatized oligonucleotides have been synthesized via phos-
phoramidite chemistry.
INTRODUCTION
Since nucleic acids play essential roles in fundamental biological pro-
cesses, the determination of 3-D structures of functional RNAs, and RNA-
protein and DNA-protein complexes by X-ray crystallography has recently
received tremendous attention174. The determination of phase in nucleic acid
*Corresponding author.
1723
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1724
CARRASCO ET AL.
X-ray crystallography is a long-standing problem. We endeavor to introduce
heavy atoms as anomalous scatterers into nucleic acids to facilitate phase
determination by the Multiwavelength Anomalous Dispersion (MAD)
technique. As selenium and oxygen are from the same family in the periodic
table, oxygen in nucleotides can be substituted with selenium. There are a
variety of oxygen atoms positioned in different chemical environments in
nucleotides, thus, selective replacement of oxygen with selenium may avoid
structural perturbation, which can not always be avoided5 in oligonucleotide
structure determination by X-ray crystallography using halogenated
nucleotides6. In addition, based on a rudimentary calculation on X-ray
phasing power of a selenium scatterer, one selenium atom can enable phase
determination for RNA or DNA up to 30 nucleotides7. Furthermore, sele-
nium substitution has proven to be successful for MAD phasing and struc-
tural determination of proteins bearing selenomethionine8710. Therefore,
selenium replacement of oxygen in nucleotides is expected to provide an ideal
scattering center, which may significantly advance 3-D structural determi-
nation of nucleic acids. In order to test this novel strategy for crystal struc-
ture determination, we have synthesized oligonucleotides containing
selenium at the 50-terminus (1, Fig. 1) by incorporating the building blocks
(2a and 2b), where the 50-oxygen is substituted by selenium. As selenols are
sensitive to air oxidation, 1 and 2b are protected as methyl selenides. The
hydrophobic nature of the methselenol functionality on 1 may enhance
strand-strand stacking interaction in crystal lattice, which may assist crys-
tallization.
Figure 1. OTBDMS: tert-butyldimethylsilyloxyl.

SELENIUM-DERIVATIZED NUCLEOSIDES
RESULTS AND DISCUSSION
1725
Incorporation of the selenium functionality is commonly done by
nucleophilic substitution chemistry in ethanol or DMF solvent using sodium
selenide made by NaBH4 reduction of selenium metal11713. However, this
conventional approach proved unsatisfactory in the case of acyl protected
nucleosides, given the fact the strong base in the nucleophilic substitution
induces removal of the acyl protecting groups on the nucleobases, along with
salt formation. To overcome this problem, we have developed the two-phase
system (H2O-toluene) to incorporate selenium using a phase transfer catalyst.
To our knowledge, this is the first example of selenide alkylation using a
phase transfer method. The half-time of the nucleophilic reaction was less
than 10 min. when Ms- (mesyl) and Br- groups were used as the leaving
groups and sodium selenide was used as the nucleophile. As this nucleophilic
substitution was fast in the organic phase, the selenide anions transferred into
the organic phase did not cause removal of the acyl groups from the
nucleosides. For C, A, and G nucleosides with acyl protection, the two-phase
reactions were conducted at pH 814, which avoid the base deprotection.
These reactions assisted by the phase transfer catalyst are fast, easy to
workup, and give high yields (usually higher than 90% yields after pur-
ification by silica gel chromatography).
The 50-hydroxy groups of partially protected nucleosides 3 (T, U, C, A,
and G) were activated for nucleophilic substitution with the leaving groups,
Br-, Ts- (tosyl), and Ms- (Scheme 1). Compound 4a was synthesized by the
Mitsunobu reaction15, and 4b and 4c (T, C, A, and G) were synthesized by
standard procedures16. In order to introduce the selenium functionality, we
initially attempted to displace the leaving groups (Br, Ms, or Ts) with sodium
selenide (Na2Se), which was generated by reduction of selenium metal with
Scheme 1. (a) CBr4=Ph3P=DIAD=THF. (b) Ts-Cl=Py. (c) Ms-Cl=TEA=THF. (d) Na2Se, (e)
Na2Se2, or (f) NaSeCH3 plus H2O=Toluene and Tetrahexylammonium Hydrogen Sulfate.
TBDMS: tert-butyldimethylsilyl.

1726
CARRASCO ET AL.
NaBH417. Because of side reactions and poor solubility of the inorganic
sodium selenide salt in organic solvents, it was difficult to develop a satis-
factory procedure for this substitution reaction in organic solvents or in
aqueous solvents, or even in mixed solvents. Finally, a two-phase system
(H2O-toluene) was developed for this substitution using a phase-transfer
catalyst (a quaternary ammonium ion).
A phase-transfer catalyst (tetrahexyl-ammonium hydrogen sulfate) was
used to shuttle the selenide anions from the aqueous phase to the organic
phase where the reaction took place. As the selenide ions are not solvated and
are highly reactive in the organic phase, when Na2Se was used as a nucleo-
phile, the nucleophilic reaction did not stop at the selenol, which was further
alkylated by another alkylating molecule, forming dialkylated product (5a).
Attempts to prevent formation of the dialkylation product by increasing the
amount of the catalyst were not successful. When a Ts- group (in case of 4b)
was applied as the leaving group instead of Br- and Ms- groups, the sub-
stitution reaction was slowed down. Nevertheless, the formation of dialky-
lation product was still observed.
The disadvantage of undesired dialkylation reaction was turned into an
advantage by using sodium diselenide (Na2Se2) instead of sodium selenide.
Sodium diselenide was prepared by fully reducing selenium metal to sodium
selenide with 0.3 eq. of NaBH4, then adding another equivalent of selenium
metal to the sodium selenide solution. The phase transfer catalyst shuttled
sodium diselenide into the organic phase, where sodium diselenide was dia-
lkylated. The dialkyl diselenide compound (5b) was stable, and reduction of
the diselenide gave the corresponding selenol in quantitative yield. As the
selenol was not stable in air, it was oxidized to the diselenide again. There-
fore, the freshly prepared selenol was used for conducting the next trans-
formation. A selenol can also be permanently protected with a stable
protecting group, such as a methyl group; this protection was achieved by
treatment with methyl iodide. As a permanent protection of the hydroseleno
group was desired in synthesis of 1 and 2b, sodium methyl selenide (prepared
by reduction of dimethyl diselenide with NaBH4) was used as the nucleophile
to react with 4c (T, C, A, and G) using the phase transfer catalyst. Although
the aqueous phase was basic, this two-phase system has completely prevented
the hydrolysis of the protecting acyl groups on A, C and G during the
reaction, which were otherwise hydrolyzed in the basic medium.
We have prepared selenium-labeled phosphoramidites 2b and oligo-
nucleotides 1 using standard phosphoramidite procedures18. The MS data
of the Se-dinucleotide (50-Se-TT) in Fig. 2A shows the selenium satellite
isotopic peaks of the molecular anion generated by the electrospray
experiment, which is consistent with the structure. The MS data of the Se-
hexanucleotide (50-Se-TGCGCA-30) shows the molecular anions carrying
from one to three negative charges (Fig. 2B); dimerization of the self-
complementary sequence carrying three charges is also revealed by the

SELENIUM-DERIVATIZED NUCLEOSIDES
1727
Figure 2. MS Spectra of Se-Oligonucleotides.
peak (1245.0 m=z). Crystallization of these oligonucleotides (the dimer and
the hexamer) for X-ray crystallography is under way. The successful
synthesis of the Se-containing oligonucleotides indicates that the selenium
functionality is compatible with the phosphoramidite solid-phase synthesis,
and large-scale Se-containing oligonucleotides can be prepared for struc-
ture determination.
EXPERIMENTAL SECTION
1-[(2R, 4S, 5R)-4-tert-butyldimethylsilyloxy-5-bromomethyl-tetrahydro-
furan-2-yl]-thymidine (4a). 3-T (261.1 mg, 0.733 mmol) and Ph3P
(577.5 mg, 2.2 mmol, 3 eq.) were placed in a 25-mL round-bottom flask
and dried on high vacuum for 1 hr THF (7.33 mL, final conc. 0.1 M), TEA
(614 mL, 6 eq.), and CBr4 (729.72 mg, 2.2 mmol, 3 eq.) were then added

1728
CARRASCO ET AL.
sequentially. The reaction mixture was stirred at RT under dry argon. The
reaction was completed after 15 min as indicated by silica gel TLC (5%
MeOH=CH2Cl2Rf ¼ 0.46). MeOH (0.5 mL) was then added to consume any
excess reagent, and the reaction mixture was stirred for another 15 min. All
the solvents were removed by rotary evaporation under reduced pressure at
30 ꢀC. The crude product was then dissolved in EtOAc, the salt was removed
by filtration, and the solvent was evaporated. The residue was directly
applied to a silica gel column (25 g of silica gel), and the column was
eluded with EtOAc=Hexane (3:7). This solution precipitated the majority
of triphenylphosphoxide, which facilitated this purification. The fractions
containing the pure product were combined and evaporated under reduced
pressure, and the resultant product was dried on high vacuum overnight to
give a brownish foamy product (298 mg, 97% yield).
1H-NMR (CDCl3) d:0.11 [(s, 6H, (CH3)2Si], 0.90 (s, 9H, t-Bu), 1.94 (s,
3H, 5-CH3), 2.1172.39 (m, 2H, 30-H), 3.5873.72 (m, 2H, 500-H), 4.074.13 (m,
1H, 50-H), 4.3574.48 (m, 1H, 30-H), 6.29 (t, J ¼ 6.75 Hz, 1H, 20-H), 7.45 (s,
1H, 6-H), 9.6579.78 (b, 1H, NH, exchangeable by D2O).
13C-NMR (CDCl3) d: 12.60 (5-CH3), 17.81 (CH3-Si), 25.81 [(CH3)3C],
00
33.03 (C5 ), 40.52 (C3 ), 73.13 (C5 ), 84.60 (C4 ), 84.76 (C2 ), 111.20 (C5),
0
0
0
0
135.58 (C6), 150.35 (C2), 163.95 (C4).
IR (KBr): 3162, 3040, 2951, 2929, 2857, 1692, 1470, 1426, 1276, 1198,
1054, 993, 904, 838, 782, 671, 561 cmÀ1.
UV (in acetonitrile): 263.8 nm.
N6-Benzoyl-1-[(2R, 4S, 5R)-4-tert-butyldimethylsilyloxy-5-methanesul-
fonyl-methyl-tetrahydrofuran-2-yl]-adenine (4c-A). 3-A (54.0 mg, 0.115 mmol)
was placed in 10-mL flask and dried on high vacuum for 2 h. THF (2.3 mL)
and TEA (47 mL, 0.345 mmol, 3 eq) were then added, and the flask was placed
on an ice-water bath and kept under dry argon. Methanesulfonyl chloride
(13 mL, 0.17 mmol, 1.5 eq) was added and the reaction was completed in
15 min. (silica gel TLC in 5% MeOH=CH2Cl2, 4c-A Rf ¼ 0.35, 3-A
Rf ¼ 0.30). MeOH (1 mL) was added to consume the excess reagent and
the reaction was stirred for another 15 min. The solvents were removed by
rotary evaporation at 40 ꢀC; the residue was dissolved in EtOAc (15 mL), and
the solution was filtered. The filtrate was then evaporated, and the residue
was purified on silica gel G60 column (gradient, from 0 to 5%
MeOH=CH2Cl2). The collected fractions were evaporated under reduced
pressure and dried on high vacuum overnight. A colorless foamy product
(4c-A) was obtained (60 mg, 98% yield).
1H-NMR (CDCl3). d: 0.13 [s, 6H, (CH3)2Si], 0.92 (s, 9H, t-Bu), 2.45-
2.55 (m, 2H, 30-H), 2.98 (s, 3H, CH3SO3 ), 4.20-4.26 (m, 1H, 50-H), 4.4074.52
(m, 2H, 500-H), 4.70-4.78 (m, 1H, 40-H), 6.4576.53 (t, J ¼ 6.6 Hz, 1H, 20-H),
7.49-7.65 (m, 3H, Ar), 8.0178.08 (m, 2H, Ar), 8.20 (s, 1H, 8-H), 8.80 (s, 1H,
2-H), 8.9579.20 (b, 1H, NH, exchangeable with D2O).

SELENIUM-DERIVATIZED NUCLEOSIDES
1729
Mass spectrum. The molecular weight of 4c-A is 547. Electrospray
experiment showed molecular peaks at 548 [M þ H]þ, 570 [M þ Na]þ.
N2-isoButyryl-1-[(2R, 4S, 5R)-4-tert-butyldimethylsilyloxy-5-methane-
sulfonyl-methyl-tetrahydrofuran-2-yl]-guanine (4c-G). The same procedure
for 4c-A was used to prepare 4c-G. The mesylation reaction was complete
in 1.5 hours. After the column chromatography over silica gel, 4c-G was
obtained in 94% yield.
1H-NMR (CDCl3) d: 0.15 [s, 6H, (CH3)2Si], 0.93 (s, 9H, t-Bu), 1.25 [s,
6H, (CH3)2C], 2.3572.64 (m, 2H, 30-H), 2.76 [sept, J ¼ 6.9 Hz, 1H,
CH(CH3)2], 3.07 (s, 3H, CH3-SO3), 4.1274.25 (m, 1H, 50-H), 4.3174.52 (m,
1H, 40-H), 4.5874.72 (m, 2H, 500-H), 6.21 (dd, J ¼ 6.0, 6.8 Hz, 1H, 20-H), 7.73
(s, 1H, 8-H), 8.87-8.95 (br, 1H, NH, exchangeable by D2O).
13C-NMR (CDCl3) d: 17.84 (CH3-Si), 18.82 [(CH(CH3)2)], 25.60
0
00
[(CH3)3C], 27.59 (CH3SO3), 36.49 [(CH(CH3)2)], 41.46 (C3 ), 66.35 (C5 ),
0
0
0
71.36 (C5 ), 86.81 (C4 ), 89.27 (C2 ), 122.48 (C5), 138.47 (C8), 146.89 (C4)
147.36 (C2), 155.86 (C6).
UV (in acetonitrile): 266.8 nm.
1-[(2R, 4S, 5S)-4-tert-butyldimethylsilyloxy-5-diselenomethyl-tetrahy-
drofuran-2-yl]-thymidine (5b). Sodium borohydride (45.1 mg, 1.2 mmol)
dissolved in water (1.2 mL) was added to a 25-mL flask containing a
suspension of selenium metal powder (94.3 mg, 1.19 mmol) in water
(1.2 mL). The reaction was placed in an ice-bath for the first few minutes
to slow down the reaction; the reaction mixture was later stirred at room
temperature under argon. After the vigorous reaction had subsided
(approximately 10 min), additional selenium metal powder (94.3 mg,
1.19 mmol) was added. The mixture was stirred for another 10 min and
then warmed on a steam bath for 5 min to completely dissolve all the
selenium and to decompose the excess NaBH4. The color of the solution
was brownish red and its pH was about 10711. This aqueous solution
was injected into a 25-mL flask containing 4a (100 mg, 0.239 mmol),
tetrahexylammonium hydrogen sulfate (10.7 mg, 0.0239 mmol, 0.1 eq.), and
toluene (4.8 mL). The reaction was closely monitored by silica gel TLC (5%
MeOH=CH2Cl2, product Rf ¼ 0.33). After 45 min the reaction was complete.
A current of air was then passed through the reaction mixture to oxidize any
excess of sodium diselenide to selenium metal, which precipitated.
The crude product mixture was centrifuged to remove the precipitated
selenium metal, followed by extraction twice with toluene (15 mL), and the
respective organic layers were combined. This organic phase was then washed
with NaHCO3 (15 mL, sat.) and NaCl (15 mL, sat.). The resultant yellowish
organic phase was dried over anhydrous mgSO4 for 30 min, the solution was
filtered, and the solvents were removed by rotary evaporation under reduced
pressure at 40 ꢀC. The crude residue was then dissolved in CH2Cl2, and the

1730
CARRASCO ET AL.
solution was loaded onto a silica gel column (6.0 g silica gel). The column was
eluted with CH2Cl2 (50 mL) followed by a stepwise gradient of MeOH (073%).
The fractions containing the product were combined and the solvents were
removed by rotary evaporation at 30 ꢀC. After drying on high vacuum over-
night, 96 mg of the yellowish diselenide product (5b) was obtained (96% yield).
1H-NMR (CDCl3) d: 0.11 [s, 6H, (CH3) Si], 0.93 (s, 9H, t-Bu), 1.94 (s,
2
3H, 5-CH3), 2.25-2.4 (m, 2H, 30-H), 3.33-3.36 (m, 2H, 500-H), 4.0374.12 (m,
1H, 50-H), 4.2874.35 (m, 1H, 40-H) 6.19 (t, J ¼ 6.6 Hz, 1H, 20-H) 7.25 (s, 1H,
6-H). 8.8078.92 (b, 1H, NH, exchangeable by D2O).
13C-NMR (CDCl3) d: 12.66 (CH3-Si), 17.92 [(CH3)3C], 25.71 (5-CH3),
0
00
0
0
0
40.41 (C3 ), 52.79 (C5 ), 74.14 (C5 ), 85.38 (C4 ), 86.04 (C2 ), 111.18 (C5),
135.72 (C6), 150.16 (C2), 164.10 (C4).
IR (KBr): 3428, 3179, 3057, 2962, 2934, 2862, 2363, 1703, 1476, 1370,
1281, 1204, 1104, 838, 782, 666 cmÀ1.
UV (in acetonitrile): 264.4 nm.
N6-Benzoyl-1-[(2R, 4S, 5R)-4-tert-butyldimethylsilyloxy-5-methselenome-
thyl-tetrahydrofuran-2-yl]-adenine (5d-A). NaBH4 (20.0 mg, 0.525 mmol)
was placed in 10mL-round flask under nitrogen. Water (1.5 mL) and
dimethyl diselenide (17.0 mL, 0.175 mmol) were sequentially and slowly
injected into the flask. Vigorous stirring helped to dissolve dimethyl
diselenide completely, forming a colorless homogeneous solution after
5710 min; the pH of the solution was higher than 11. Since high pH
caused the hydrolysis of the protecting benzoyl group on adenine base,
and pH 7.0 or lower made the following selenide substitution very slow, the
pH was adjusted to 8.0 by adding dilute HCl dropwise. 4c-A (19.2 mg,
0.0350 mmol) and tetrahexylammonium hydrogen sulfate (0.5 mg) dissolved
in toluene (0.7 mL) were then added to the sodium methylselenide solution
(pH 8.0) described above, and the two-phase mixture (toluene and water) was
stirred under nitrogen. The reaction was complete after 5 h, forming 5d-A
(silica gel TLC, 5% MeOH=CH2Cl2, Rf ¼ 0.52). Longer reaction time caused
slowhydrolysis of the benzoyl group (the hydrolyzed product R ¼ 0.33 on
f
TLC, 5% MeOH=CH2Cl2). The organic phase was removed and the aqueous
phase was extracted twice with EtOAc; the combined organic phase was
washed twice with saturated NaCl solution. The solvents were removed
by rotary evaporation under reduced pressure at 40 ꢀC. The crude product
was dissolved in CH2Cl2 and loaded on a silica gel TLC plate (5%
MeOH=CH2Cl2). Colorless product was recovered from this purification
(18.2 mg, 95% yield).
1H-NMR (CDCl3). d: 0.12 [s, 6H, (CH3)2Si], 0.92 (s, 9H, t-Bu), 2.02 (s, 3H,
CH3Se), 2.4372.53 (m, 1H, 30-H), 2.7873.00 (m, 3H, 30-H and 500-H), 4.1574.23
(m, 1H, 50-H), 4.5574.63 (m, 1H, 40-H), 6.4276.50 (t, J ¼ 6.6 Hz, 1H, 20-H),
7.4877.57 (m, 2H, Ar), 7.5777.65 (m, 1H, Ar), 8.0078.07 (m, 2H, Ar), 8.27
(s, 1H, 8-H), 8.80 (s, 1H, 2-H), 8.9479.00 (b, 1H, NH, exchangeable by D2O).

SELENIUM-DERIVATIZED NUCLEOSIDES
1731
The molecular weight of 5d-A (C24H33N5O2SiSe) is 547 with adjustment
for 80Se isotope [average atomic weight of Se is 79, including 76 (9%), 77
(7%), 78 (23%), 80 (49%), 82 (9.2%)]. The molecular peaks are: 546
[M(78Se)þH]þ, 548 [M(80Se)þH]þ, 550 [M(82Se)þH]þ, 568 [M(78Se)þNa]þ,
570 [M(80Se)þNa]þ, 572 [M(82Se)þNa]þ.
1-[(2R,4S,5R)-4-tert-butyldimethylsilyloxy-5-methselenomethyl-tetra-
hydro-furan-2-yl]-thymidine (5d-T)
Method 1. NaBH4 (63 mg, 1.65 mmol) was dissolved in 1.5 ml water
and sealed in a 10-mL flask under nitrogen. Dimethyl diselenide (54 mL,
0.55 mmol) was slowly injected into the flask. Addition of 0.2 mL of
ethanol with vigorous stirring helped to dissolve dimethyl diselenide
completely; a colorless homogeneous solution was formed in 5 min. The
solution of 4c-T (47.7 mg, 0.110 mmol) and tetrahexylammonium hydrogen
sulfate (1 mg) in toluene (1.1 mL) was then added to the sodium methyl
selenide solution described above. The two-phase mixture (toluene and
water) was stirred under nitrogen. The reaction was complete in 3 h as
indicated silica gel TLC (5% MeOH=CH2Cl2, Rf ¼ 0.40). The organic
phase was removed, the aqueous phase was extracted twice with EtOAc
(10 mL each time), and the combined organic phase was washed twice
with saturated NaCl solution (10 mL each time). The solvents were
removed by rotary evaporation under reduced pressure at 40 ꢀC. The
crude product was purified on TLC (5% MeOH=CH2Cl2). The pure
product (5d-T) was dried on high vacuum overnight to afford 44.2 mg
(93% yield).
Method 2. The title compound was also made by reduction of the dis-
elenide compound 5b with NaBH4 in EtOH. After the yellowsolution of the
diselenide turned into colorless, indicating production of the selenol (5c)
(approximately 5 min), CH3I was added to protect 5c. This approach gave
quantitative yield.
1H-NMR (CDCl3) d: 0.05 [s, 6H, (CH3)2Si], 0.88 (s, 9H, t-Bu), 1.94 (s,
3H, 5-CH3), 2.08 (s, 3H, CH3-Se), 2.0572.18 and 2.2872.40 (2m, 2H, 30-H),
2.7872.93 (m, 2H, 500-H), 4.0274.09 (m, 1H, 50-H), 4.2874.38 (m, 1H, 40-H),
6.26 (t, J ¼ 6.5 Hz, 1H, 20-H), 7.42 (s, 1H, 6-H), 8.9579.06 (b, 1H, NH,
exchangeable in D2O).
13C-NMR (CDCl3) d: 5.66 (CH3-Se), 12.55 (5-CH3), 17.68 (CH3-Si),
00
25.68 [(CH3)3C], 27.63 (C5 ), 40.63 (C3 ), 73.94 (C5 ), 84.57 (C4 ), 85.60 (C2 ),
0
0
0
0
111.11 (C5), 135.59 (C6), 150.18 (C2), 163.69 (C4).
The MS spectrum of 5d-T is shown in Figure 3 (electrospray, positive
ion experiment). The molecular weight (C17H30N2O4SiSe) is 434 with
adjustment for 80Se isotope. Because of binding of H or Na ions, two sets of
molecular peaks are observed, in which all selenium isotopic peaks [Se 76
(9%), 77 (7%), 78 (23%), 80 (49%), 82 (9.2%)] are also observed: 431 [M
(76Se)þH]þ, 432 [M (77Se)þH]þ, 433 [M (78Se)þH]þ, 435 [M (80Se)þH]þ, 437

1732
CARRASCO ET AL.
Figure 3. MS of 1-[(2R, 4S, 5R)-4-tert-butyldimethylsilyloxy-5-methselenomethyl-tetra-
hydro-furan-2-yl]-thymidine (5d-T).
[M (82Se)þH]þ, 453 [M (76Se)þNa]þ, 454 [M (77Se)þNa]þ, 455 [M
(78Se)þNa]þ, 457 [M (80Se)þNa]þ, and 459 [M (82Se)þNa]þ.
IR (KBr): 3157, 3034, 2960, 2940, 2862, 2363, 1698, 1470, 1426, 1370,
1276, 1198, 1120, 1089, 1049, 827, 777, 682, 621 cmÀ1.
UV (in acetonitrile): 265.2 nm.
1-[(2R, 4S, 5R)-4-hydroxy-5-methselenomethyl-tetrahydrofuran-2-yl]-
thymidine (5d-T-OH). Following the standard procedure, compound 5d-T
(50 mg, 0.115 mmol) was dissolved in THF (345 mL), and tert-butyl
Figure 4. 77Se-NMR of 1-[(2R, 4S, 5R)-4-hydroxy-5-methseleno-methyl-tetrahydrofuran-2-
yl]-thymidine in CDCl3; reference (dibenzyl diselenide, 133.25 ppm).

SELENIUM-DERIVATIZED NUCLEOSIDES
1733
ammonium fluoride 1 M in THF (230 mL, 2 eq.) was added. The deprotection
reaction was complete in hours (monitored by TLC, 7.5%
2
MeOH=CH2Cl2). The product was purified on TLC to give quantitative
yield, and the structure of this product was confirmed by spectroscopy
analysis, including 77Se-NMR (Fig. 4).
1H-NMR (CD3OD=CDCl3 ¼ 1:1) d: 1.93 (s, 3H, 5-CH3), 2.06 (s, 3H,
CH3-Se), 2.1572.28 and 2.3272.46 (2m, 2H, 30-H), 2.8272.96 (m, 2H, 500-H),
4.0574.15 (m, 1H, 50-H), 4.2874.38 (m, 1H, 40-H), 6.26 (t, J ¼ 6.7 Hz, 1H, 20-
H), 7.49 (s, 1H, 6-H), 8.9579.06 (b, 1H, NH, exchangeable in D2O).
13
00
C-NMR (CDCl3) d: 5.31 (CH3-Se), 12.11 (5-CH3), 27.49 (C5 ), 39.74
0
0
0
0
(C3 ), 72.88 (C5 ), 84.21 (C4 ), 84.14 (C2 ), 110.89 (C5), 135.70 (C6), 150.44
(C2), 163.69 (C4).
As 77Se NMR active (MI ¼ 1=2), a Se-NMR was done (Fig. 4). 77Se-
NMR (CDCl3) d: 362.55 ppm (reference: dibenzyl diselenide, 133.25 ppm)
IR (KBr): 3473, 3167, 3095, 2960, 2923, 2812, 2679, 1703, 1476, 1410,
1259, 1071, 1015, 950, 888, 816, 632, 570 cmÀ1.
UV (in acetonitrile): 265.0 nm.
ACKNOWLEDGMENT
This work was supported by PSC-CUNY Research Award (#69674) and New
Faculty Awards. We thank Dr. Hsing Wang for collecting 77Se-MNR data and Dr.
Clifford E. Soll for providing MS data. NC thanks NYC LSAMP and National
Science Foundation for the fellowships; DG thanks Pfizer for an undergraduate
student summer fellowship. We also thank Drs. Alexander Greer, Paul Haberfield,
and Richard Maliozzo for commenting on the manuscript.
REFERENCES
1. Nissen, P.; Hansen, J.; Ban, N.; Moore, P.B.; Steitz, T.A. The Structural Basis
of Ribosome Activity in Peptide Bond Synthesis. Science 2000, 289, 9207930.
2. Ban, N.; Nissen, P.; Hansen, J.; Capel, M.; Moore, P.B.; Steitz, T.A. Placement
of Protein and RNA Structures into a 5 A-Resolution Map of the 50S Riboso-
mal Subunit. Nature 1999, 400, 8417847.
3. Golden, B.L.; Gooding, A.R.; Podell, E.; Cech, T.R. A Preorganized Active
Site in the Crystal Structure of the Tetrahymena Ribozyme. Science 1998,
282, 2597264.
4. Cate, J.H.; Gooding, A.R.; Podell, E.; Zhou, K.; Golden, B.L.; Szewczak,
A.A.; Kundrot, C.E.; Cech, T.R.; Doudna, J.A. RNA Tertiary Structure Med-
iation by Adenosine Platforms. Science 1996, 273, 169671699.
5. a). Kim, R.; Holbrook, E.L.; Jancarik, J.; Pandit, J.; Weng X.; Bohm, A.; Kim,
S.-H. High-Resolution Crystals and Preliminary X-ray Diffraction Studies of a
Catalytic RNA. Acta Cryst. 1994, D50, 2907292; b). S. Holbrook, Personal
Communication; c). M. Egli, Personal Communication.

1734
CARRASCO ET AL.
6. a). Usman, N.; Egli, M.; Rich, A. Large Scale Chemical Synthesis, Purification
and Crystallization of RNA-DNA Chimeras. Nucleic Acids Res., 1992, 20,
669576699; b). Pley, H.W.; Flaherty, K.M.; Mckay, D.B. Three-dimensional
Structure of a Hammerhead Ribozyme. Nature 1994, 372, 68774; c). Naber,
N.; Matuska, M.; Sablin, E.P.; Pate E.; Cooke, R. A Novel Adenosine
Triphosphate Analog with a Heavy Atom to Target the Nucleotide Binding
Site of Proteins. Protein Science 1995, 4, 182471831.
7. Hendrickson, W.A; Ogata, C.M. Phase Determination from Multiwavenlength
Anomalous Diffraction Measurements. Methods in Enzymology, Part A, 1997,
276, 4947523.
8. Hendrickson, W.A.; Pahler, A.; Smith, J.L.; Satow, Y.; Merritt, E.A.; Phizack-
erley, R.P. Crystal Structure of Core Streptavidin Determined from Multiwa-
velength Anomalous Diffraction of Synchrotron Radiation. Proc. Natl.
Acad. Sci. USA 1989, 86, 219072194.
9. Yang, W.; Hendrickson, W.A.; Crouch, R.J.; Satow, Y. Structure of Ribonu-
clease H Phased at 2 A Resolution by MAD Analysis of the Selenomethionyl
Protein. Science 1990, 249, 139871405.
10. Ferre-D’Amare, A.R.; Zhou, Kaihong; Doudna, J.A. Crystal Structure of a
Hepatitis Delta Virus Ribozyme. Nature 1998, 395, 5677574.
11. Hale, K.J.; Manaviazar, S. Novel Pyranoid Glycals Derived from D-Fructose.
Tetrahedron Lett. 1994, 35, 887378876.
12. Kawashima, E.; Toyama, K.; Ohshima, K.; Kainosho, M.; Kyogoku, Y.;
Ishido, Y. Novel Synthesis of 20-deoxy[50-2H]Ribonucleosides Derivatives
from 50-O-Ac-20-deoxy-50-PhSe-ribonucleoside Derivatives. Tetrahedron Lett.
1995, 36, 669976700.
13. Andreadou, I.; Menge, W.M.P.B.; Commandeur, J.N.M.; Worthington, E.A.;
Vermeulen, N.P.E. Synthesis of Novel Se-Substituted Selenocysteine Deriva-
tives as Potential Kidney Selective Prodrugs of Biologically Active Selenol
Compounds: Evaluation of Kinetics of -Elimination Reactions in Rat Renal
Cytosol. J. Med. Chem. 1996, 39, 204072046.
14. Mautner, H.C. The Synthesis and Properties of Some Selenopurines and Sele-
nopyrimidines. J. Am. Chem. Soc. 1956, 78, 529275294.
15. Richert, C.; Roughton, A.L.; Benner, A.S. Nonionic Analogs of RNA with
Dimethylene Sulfone Bridges. J. Am. Chem. Soc. 1996, 118, 451874531.
16. Huang, Z.; Schneider, K.C.; Benner, S.A. Building Blocks for Oligonucleotide
Analogues with Dimethylene Sulfide, Sulfoxide, and Sulfone Groups Replacing
Phosphodiester Linkages. J. Org. Chem. 1991, 56, 386973882.
17. Jakiwczyk, O.M.; Kristoff, E.M.; McPhee, D. Convenient Preparation of Dia-
lkyl Diselenides: A Large Scale Synthesis of bis-(2-hydroxyethyl) Diselenide.
Synthetic Communication 1993, 23, 1957199.
18. Tang, X.-Q.; Liao, X.; Piccirilli, J.A. 20-C-Branched Ribonucleosides: Synthesis
of the Phosphoramidite Derivatives of 20-C-Methylcytidine and Their Incor-
poration into Oligonucleotides. J. Org. Chem. 1999, 64, 7477754.
Received December 30, 2000
Accepted April 6, 2001