Chemical Synthesis of Selenium-Modified Oligoribonucleotides and Their Enzymatic Ligation Leading to an U6 SnRNA Stem-Loop Segment

Article abstract of DOI:10.1021/ja038481k

The derivatization of nucleic acids with selenium is highly promising to facilitate nucleic acids structure determination by X-ray crystallography using the multiwavelength anomalous dispersion (MAD) technique. The foundation for such an approach has been laid by Huang, Egli, and co-workers and was exemplified on small DNA duplexes. Here, we present a comprehensive study on the preparation of RNAs containing 2′-Se-methylpyrimidine nucleoside labels. This includes the synthesis of a novel 2′-Semethylcytidine phosphoramidite 11 and its incorporation into oligoribonucleotides by solid-phase synthesis. Deprotection of the oligonucleotides is achieved in the presence of millimolar amounts of threo-1,4-dimercapto-2,3-butandiol (DTT). With this additive, oxidation products and follow-up side-products are suppressed and acceptable HPLC traces of the crude material are obtained, so far tested for sequences of up to 22-mers. Moreover, an extensive investigation on the enzymatic ligation of the selenium-containing oligoribonucleotides demonstrates the high flexibility of the selenium approach. Our target sequence, an U6 snRNA stem-loop motif comprising all naturally occurring nucleoside modifications beside the Selabel is achieved by ligation using T4 RNA ligase.

Full text of DOI:10.1021/ja038481k

Published on Web 01/09/2004
Chemical Synthesis of Selenium-Modified
Oligoribonucleotides and Their Enzymatic Ligation Leading to
an U6 SnRNA Stem-Loop Segment
Claudia Ho¨bartner and Ronald Micura*
Contribution from the Institute of Organic Chemistry, Leopold Franzens UniVersity, Innrain 52a,
A-6020 Innsbruck, Austria
Received September 12, 2003; E-mail: ronald.micura@uibk.ac.at
Abstract: The derivatization of nucleic acids with selenium is highly promising to facilitate nucleic acids
structure determination by X-ray crystallography using the multiwavelength anomalous dispersion (MAD)
technique. The foundation for such an approach has been laid by Huang, Egli, and co-workers and was
exemplified on small DNA duplexes. Here, we present a comprehensive study on the preparation of RNAs
containing 2′-Se-methylpyrimidine nucleoside labels. This includes the synthesis of a novel 2′-Se-
methylcytidine phosphoramidite 11 and its incorporation into oligoribonucleotides by solid-phase synthesis.
Deprotection of the oligonucleotides is achieved in the presence of millimolar amounts of threo-1,4-
dimercapto-2,3-butandiol (DTT). With this additive, oxidation products and follow-up side-products are
suppressed and acceptable HPLC traces of the crude material are obtained, so far tested for sequences
of up to 22-mers. Moreover, an extensive investigation on the enzymatic ligation of the selenium-containing
oligoribonucleotides demonstrates the high flexibility of the selenium approach. Our target sequence, an
U6 snRNA stem-loop motif comprising all naturally occurring nucleoside modifications beside the Se-
label is achieved by ligation using T4 RNA ligase.
was obtained proving the principle of the concept.6 Therein,
Introduction
the 2′-Se-methylribofuranoses display C3′-endo puckers, con-
sistent with A-form geometry. Moreover, Egli and co-workers
documented the preparation and crystal structure of a Z-form
DNA duplex with a phosphoroselenoate modified backbone,
[d(CPSeGCGCG)]2.7 The phosphoroselenoate modification was
accomplished during solid-phase oligonucleotide synthesis by
replacing the standard oxidation agent with potassium seleno-
cyanide. The resulting diastereomeric mixture of phosphoro-
selenoates had to be separated by anion exchange HPLC. Only
very short sequences are therefore readily accessible. For DNA
sequences, this limitation might be overcome if nonenzymatic
template-directed ligation of oligonucleotide 3′-phosphorose-
lenoate anions to oligonucleotide 5′-iodides is considered,
according to a procedure published by Kool.8 Diastereomerically
pure products would be directly obtained.
The rapidly growing knowledge about the significance of
small RNAs in many biological processes is also accompanied
by a great demand for RNA structure analysis by X-ray
crystallography. In this context, selenium-modified RNA would
be immensely valuable in order to facilitate phasing of diffrac-
tion data. Unfortunately, little data are available with respect
to chemical preparation.9-11 Only the synthesis of a single
hexamer and a short RNA-DNA hybrid have been documented
without providing experimental details.5 Therefore, we have
High-resolution structure analysis of biomolecules is of
tremendous importance to gain insight into fundamental biologi-
cal processes. X-ray crystallography using the multiwavelength
anomalous dispersion (MAD) technique has been widely used
in recent years and accounts for the majority of all new protein
crystal structures.2 This approach implicates the use of sele-
nomethionyl-modified proteins. The selenomethionine replaces
natural methionine residues as a result of overexpression of the
target protein in the presence of selenomethionine.3,4 For MAD
experiments selenium serves as an effective anomalous scat-
tering center and greatly facilitates phase determination of
protein crystals. The first steps to apply this successful concept
in nucleic acid structure analysis have been made recently.
Huang, Egli, and co-workers reported a 2′-Se-methyl-uridine
(USe) phosphoramidite 4 that allowed for the incorporation into
small oligonucleotides by solid-phase synthesis.5 The crystal
structure of the A-form DNA duplex [d(GCGTA)USed(ACGC)]2
* To whom correspondence should be addressed. Phone: ++43 512
507 5212. Fax: ++43 512 507 2892.
(1) The following abbreviations are used: MAD, multiwavelength anomalous
dispersion; DMT, dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; TOM,
(triisopropylsilyl)oxymethyl; Pac, phenoxyacetyl; TBAF, tetrabutylammo-
nium fluoride; DMAP, 4-(dimethylamino)-pyridine; DTT, threo-1,4-
dimercapto-2,3-butandiol; ATP, adenosine triphosphate; EDTA, ethylen-
diaminetetraacetate; Tris, 2-amino-2-hydroxymethyl-1,3-propandiol; TMS,
tetramethylsilane; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; DMF,
N,N-dimethylformamide; TLC, thin-layer chromatography; MALDI-TOF,
matrix assisted laser desorption ionization time-of-flight; FT ICR ESI-MS,
Fourier transformation ion cyclotron resonance electrospray ionization mass
spectrometry.
(5) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z. J.
Am. Chem. Soc. 2002, 124, 24-25.
(6) Teplova, M.; Wilds, C. J.; Wawrzak Z.; Tereshko V.; Du Q.; Carrasco,
N.; Huang, Z.; Egli, M. Biochimie 2002, 84, 849-858.
(7) Wilds, C. J.; Pattanayek R.; Pan C.; Wawrzak Z.; Egli, M. J. Am. Chem.
Soc. 2002, 124, 14910-14916.
(2) Hendrickson, W. A.; Ogata, C. M. Methods Enzymol. 1997, 276, 494-
523.
(3) Doublie´, S. Methods Enzymol. 1997, 276, 523-530.
(4) Hendrickson, W. A. Trends Biochem. Sci. 2000, 25, 637-643.
(8) Xu, Y.; Kool E. T. J. Am. Chem. Soc. 2000, 122, 9040-9041.
9
10.1021/ja038481k CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 1141-1149
1141

A R T I C L E S
Ho¨bartner and Micura
Scheme 1. Synthesis of 2′-Se-methyl Uridine Phosphoramiditea
focused on the development of methodologies for the preparation
of selenium-modified RNAs as their availability still represents
the bottleneck for broad systematic crystallization screenings.
Here, we present a robust preparation protocol for such RNAs.
Our approach relies on the incorporation of 2′-Se-methyl
modified uridine and cytidine nucleosides, 4 and 11. To the
best of our knowledge, this is the first study that demonstrates
comprehensively the synthetic feasibility of high-purity selenium-
labeled RNA with a length of up to thirty nucleotides and in
amounts sufficient for crystallization screenings.
Results and Discussion
We started our studies on selenium-modified RNA based on
the knowledge reported on 2′-Se-methyl modified DNA.5,6
Ribose 2′-Se-methyl moieties are in principle comparable with
the naturally occurring ribose 2′-O-methyl modifications and
can therefore be considered as an appropriate replacement. These
modifications support the C3′-endo type ribose puckering
beneficial for A-form geometry. From the crystal structure of
the A-form DNA duplex [d(GCGTA)USed(ACGC)]2, it is also
known that the methylseleno moieties are directed toward the
minor groove as generally observed for 2′-O-alkylated nucleo-
sides.6 The van der Waals radius of selenium is 2.00 Å as
compared to 1.40 Å for oxygen. This may cause structural
perturbations and effect crystallization efforts. However, these
perturbations have to be considered less than those observed
for 5-bromo- or 5-iodopyrimidine oligonucleotides, which
represent the majority of probes with covalent derivatization in
nucleic acid crystallography.12-15 5-Halogen modifications can
cause disruption or severe alterations of nucleobase stacking;
they are light-sensitive and may cause decomposition of the
oligonucleotide during long-time exposure to X-ray sources.
Syntheses of 2′-Se-Methyl Pyrimidine Nucleoside Phos-
phoramidites. As a first step we intended to improve the
accessibility of 2′-Se-methyluridine phosphoramidite 4 (Scheme
1). Therefore, uridine was directly transformed into 2,2′-
anhydrouridine 1 using diphenyl carbonate in DMF.16 The 5′-
OH was then protected as dimethoxytrityl (DMT) ether to form
compound 2. Subsequent treatment with sodium methylselenide
in THF was used for ring-opening to furnish the 2′-Se-methyl
derivative 3.5 Conversion into the corresponding phosphora-
midite 4 was achieved in good yields by reaction with
2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Compared
to the procedure by Huang, key intermediate 2 was obtained
from uridine in two steps instead of five via the 5′-O-DMT-
3′-O-TBDMS functionalized intermediate.5
a (a) 1.1 equiv of diphenyl carbonate, 3.6% sodium hydrogencarbonate
in DMF, 120 °C, 4 h, 75%.16 (b) 1.1 equiv of DMT-Cl in pyridine, room
temperature, 6 h, 67%.16 (c) 2 equiv of dimethyldiselenide, 6 equiv of sodium
borohydride in THF, room temperature, 2 h, 92%.5 (d) 1.5 equiv of (2-
cyanoethyl) N,N-diisopropylchlorophosphoramidite, 10 equiv of ethyldi-
methylamine, in CH2Cl2, room temperature, 2 h, 93%.5 (e) 1 equiv of
DMAP, 4.5 equiv of bis-(4-nitrophenyl)hexandioate in pyridine/DMF, room
temperature, 24 h, 85%.
therefore decided on a strategy that involves transformation of
the nucleobase. For this, the 3′-OH of derivative 3 was protected
applying TBDMS chloride and imidazole in DMF to obtain
compound 6 (Scheme 2). Then, reaction of 6 with 2,4,6-
triisopropylbenzenesulfonyl chloride in the presence of triethyl-
amine and DMAP in dichloromethane resulted in regioselective
O4-trisylation. After workup, the trisylated derivative 7 was used
without further purification and directly converted into 8 upon
treatment with aqueous ammonium hydroxide in THF in 79%
yield over the two steps. Noteably, treatment of 8 with 7 M
NH3 in anhydrous methanol resulted in the O4-methyluridine
derivative. Acetylation of the amino function was then achieved
with acetic anhydride in pyridine to provide 9, followed by
cleavage of the 3′-O-TBDMS group with 1 M TBAF and 0.5
M acetic acid in THF. Finally, conversion of 10 into the 2′-
Se-methylcytidine phosphoramidite 11 proceeded by reaction
with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite in the
presence of ethyldimethylamine in 82% yield.
Initial attempts to synthesize the corresponding 2′-Se-meth-
ylcytidine phosphoramidite 11 by an analogous route starting
from commercially available 2,2′-anhydrocytidine failed. We
(9) Mori, K.; Boiziau, C.; Cazenave, C.; Matsukura, M.; Subasinghe, C.; Cohen,
J. S.; Broder, S.; Toulme, J. J.; Stein, C. A. Nucl. Acids Res. 1989, 17,
8207-8219.
(10) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucleotides
Nucleic Acids 2001, 20, 1723-1734.
Oligoribonucleotide Synthesis and Purification. All oligori-
bonucleotides were synthesized using nucleobase-acetylated and
2′-O-TOM protected nucleoside phosphoramidites (Table 1).17,18
The incorporation of 2′-Se-methyluridine and cytidine phos-
phoramidites was performed with standard DNA/RNA solid-
phase synthesis protocols with coupling yields higher than 98%
(11) Holloway, G. A.; Pavot C.; Scaringe, S. A.; Lu, Y., Rauchfuss, T.;
Chembiochem 2002, 3, 1061-1065.
(12) Shui, M. E.; Peek, M. E.; Lipscomb, L. A.; Gao, Q.; Ogata, C.; Roques,
B. P.; Garbay-Jaureguiberry, C.; Wilkinson, A. P.; Williams, L. D. Curr.
Med. Chem. 2000, 7, 59-71.
(13) Deng, J.; Xiong, Y.; Sundaralingam M. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 13 665-13 670.
(14) Wing, R.; Drew, H.; Takano, T.; Broka, C.; Tanaka, S.; Itakura, K.;
Dickerson, R. E. Nature 1980, 287, 755-758.
(15) Zhang, L.; Doudna, J. A. Science 2002, 295, 2084-2088.
(16) McGee, D. P. C.; Vaughn-Settle, A.; Vargeese, C.; Zhai, Y. J. Org. Chem.
1996, 61, 781-785.
(17) Micura, R. Angew. Chem., Int. Ed. Engl. 2002, 41, 2265-2267.
(18) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HelV. Chim. Acta
2001, 84, 3773-3795.
9
1142 J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004

Selenium-Modified Oligoribonucleotides
A R T I C L E S
Scheme 2. Synthesis of 2′-Se-methyl Cytidine Phosphoramiditea
Figure 1. Optimization of the deprotection of Se-derivatized oligoribo-
nucleotides. (a) HPLC trace of crude CGCGUSeGG 12 after standard
deprotection conditions for 2′-O-TOM RNAs (1, MeNH2 in EtOH/H2O, 6
h, room temperature; 2, 1 M TBAF in THF, 12 h, room temperature). Major
products were isolated and characterized individually by MALDI-TOF
MS: 12 (m/z calcd [M + H]+ 2313.4, found 2312.9(2); oxidation product
12a ([M + H]+, found 2328.9(2); for details see supporting information.
(b) HPLC trace of crude CGCGUSeGG 12 obtained by addition of 10 mM
DTT during treatment with the above-mentioned deprotection solutions.
(c) HPLC trace of crude and purified (inset) AAGCSeACACAAACC(dA)-
(dG)(dA)CGGCC 24 (50 mM DTT). Anion exchange HPLC: Dionex
DNAPac (4 × 250 mm), 80 °C, 1 mL/min, 0-40% B in 30 min (0-60 B
in 45 min for (c)); A, 25 mM Tris‚HCl, 6 M urea, pH 8.0; B, same as A
+ 0.5 M NaClO4.
a (a) 2 equiv of TBDMS-Cl, 4 equiv of imidazole in DMF, room
temperature, 18 h, 95%. (b) 1.5 equiv of 2,4,6-triisopropylbenzenesulfonyl
chloride, 10 equiv of NEt3, 0.12 equiv of DMAP in CH2Cl2, room
temperature, 1 h. (c) aqueous ammoniumhydroxide in THF, room temper-
ature, 12 h, 79% (over (b) and (c)). (d) 2.5 equiv of acetic anhydride in
pyridine, room temperature, 30 min, 94%. (e) 1 M TBAF, 0.5 M acetic
acid in THF, room temperature, 2.5 h, 87%. (f) 1.5 equiv of (2-cyanoethyl)
N,N-diisopropylchlorophosphoramidite, 10 equiv of ethyldimethylamine, in
CH2Cl2, room temperature, 2 h, 82%.
combined with varying conditions for TOM deprotection with
fluoride at different temperatures and with different reaction
times were not successful. However, we finally found that
addition of millimolar amounts of threo-1,4-dimercapto-2,3-
butandiol (DTT) to the solutions of CH3NH2 in ethanol/water
drastically improved the quality of the crude materials and also
improved reproducibility.19 This is illustrated for sequence 12,
5′-CGCGUSeGG in Figure 1. Concerning this sequence, we also
purified the major byproduct 12a that was observed when
deprotection conditions without DTT had been applied. By
MALDI-TOF mass spectrometry a [M + H]+ signal was
obtained with 16 mass units higher than expected for 12, 5′-
CGCGUSeGG, indicating that the selenium moiety was most
likely oxidized (supporting information). From the above, it is
obvious that DTT reduces the amount of oxidation products.
Oxidation of the Se moieties most likely occurs during strand
assembly when after each coupling step the phosphite triester
is transformed into the corresponding phosphate triester by
treatment with I2. To understand how DTT acts on the oxidized
according to the trityl assay (supporting information). For
phosphite oxidation, a 10 mM solution of I2 in acetonitrile/2,4,6-
collidine/water was applied for 1 min after each coupling. Even
though the 2′-Se-methyl nucleosides were placed at the 3′-end
of a sequence and therefore repeatedly exposed to oxidative
conditions, the coupling yields throughout the various syntheses
were comparable to those of non-selenium sequences. This sug-
gested that the selenium moieties are sufficiently stable during
strand assembly although oxidation products and follow-up elim-
ination products could not be completely excluded at this level.
After strand assembly, we originally encountered severe
difficulties with respect to deprotection of the selenium-
containing oligoribonucleotides. Standard conditions, such as
CH3NH2 in ethanol/water for the deprotection of acetyl and
cyanoethyl groups and cleavage from the solid support followed
by treatment with 1 M TBAF in THF to cleave the silyl ethers,
resulted for the majority of the crude products in HPLC traces
showing multiple peaks. Variation of the conditions using CH3-
NH2 in ethanol, NH3 in methanol, or aqueous ammonia
(19) Ho¨bartner, C.; Micura, R. RNA 2003, 8th Annual Meeting of the RNA
Society, July 1-July 6, 2003, Vienna, Austria; Book of Abstracts, p 323.
9
J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004 1143

A R T I C L E S
Ho¨bartner and Micura
Table 1. Solid Phase Synthesis of RNA Oligonucleotides Containing 2′-Se-Methyl Nucleosidesa
isolated yield
molecular weight
260 nm
no.
sequencea
scale, µmol
OD
nmol
calcd amu
foundb amu
12
13
14
15
16
17
18
19
20
21
22
23
24
25
5′-CGCGUSeGG-3′
5′-CGCGUSeGGC-3′
5′-pUSeGACACGCUp-3′
5′-pUGACACGCUSep-3′
5′-pUGACACSeGCUp-3′
5′-pUGACmACGCUSep-3′
5′-pUGACmACSeGCUp-3′
5′-GCSeGGCGGCGGC-3′
5′-GCGGCGGCGGUSe-3′
5′-GCSeGGCGGC-3′
5′-USeGCGGCGGCGGC-3′
5′-anthracene(heg)-GGAGCUSeCGCCSeC-3′
5′-AAGCSeCACACAAACC(dA)(dG)(dA)CGGCC-3′
5′-AAGCSeCACACAAACC(dA)(dG)(dA)CSeGGCC-3′
0.5
0.7
0.3
0.3
0.3
0.3
0.3
0.3
3
2
1
2
2
8.3
10.4
8.3
4.4
2.3
2.9
2.1
10.3
115
22.0
16.9
126
67.8
2.5
116
131
86
46
24
30
22
93
2312.4
2617.6
3051.7
3051.7
3051.7
3065.7
3065.7
3652.2
3653.2
2656.6
3958.3
4168.6
7057.3
7134.3
2311.9
2618.6
3054.5
3053.3
3054.4
3064.1
3067.3
3655.2
3655.7
2657.8
3959.1
4168.2
7059.7
7136.6
1000
270
140
270
10
0.3
a USe, 2′-Se-methyluridine; CSe, 2′-Se-methylcytidine; Cm, 2′-O-methylcytidine; dA, 2′-O-deoxyadenosine; dG, 2′-O-deoxyguanosine; heg, hexaethyleneglycol.
MALDI-TOF MS; molecular weights detected as [M + H]+; [M + Na]+ for sequence 23.
b
Figure 2. (a) Secondary structure of rat spliceosomal U6 snRNA.21 (b) U2/U6 snRNA interaction at the catalytic center of the spliceosome, maintaining the
boxed stem-loop segment of nucleotides 57-78. Am, 2′-O-methyladenosine; Cm, 2′-O-methylcytidine; Gm, 2′-O-methylguanosine; Um, 2′-O-methyluridine;
m6A, N6-methyladenosine; m2G, N2-methylguanosine; Ψ, pseudouridine.
products, such as 12a, is issue of our ongoing work. In this
context, we note that the use of a 1.1 M solution of tert-
butylhydroperoxide in THF instead of treatment with I2 was
tested and yielded products of comparable quality.
We further note that after evaporation of the basic deprotec-
tion solution, the nonvolatile DTT was not separated from the
crude product but directly mixed with the TBAF/THF solutions
to cleave the TOM groups. DTT was removed after the two-
step deprotection procedure together with salts by size exclusion
chromatography on a Sephadex column.
with up to 22 nucleotides and containing up to two methylseleno
nucleosides. The molecular weights of the purified sequences
have been confirmed by MALDI-TOF mass spectrometry
(supporting information). Sequences 19-24 have been prepared
in larger amounts to enable crystallization screenings that are
currently in progress in the laboratory of our collaborators.
Enzymatic Ligation of Oligoribonucleotides Containing
2′-Se-Methyl Nucleosides. We intended to prove the acces-
sibility also of larger seleno-RNAs by a comprehensive enzy-
matic ligation study using T4 RNA ligase. T4 RNA ligase
possesses the ability to join the 3′-hydroxyl and the 5′-phosphate
termini of individual ribonucleic acid strands.20 As target
sequence we have chosen a rat spliceosomal U6 snRNA stem-
loop motif (Figure 2, box).21 This motif comprising the
Purification of the crude deprotected oligoribonucleotides was
then accomplished by anion exchange chromatography on
semipreparative columns under strong denaturating conditions
(6 M urea, 80 °C). Table 1 shows a series of oligoribonucleotides
9
1144 J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004

Selenium-Modified Oligoribonucleotides
A R T I C L E S
nonmodified sequences to optimize the general reaction and
buffer conditions. Strand concentrations of 40-120 µM and 0.4
U/µL T4 RNA ligase in Tris buffer (pH 7.8) in the presence of
MgCl2, DTT, and ATP at 37 °C turned out to be appropriate.
Good overall ligation yields were, however, only obtained if
the donor substrate (providing the 5′-phosphate terminus) was
represented by a 5′,3′-bisphosphate oligoribonucleotide. When
5′-phosphate oligoribonucleotides with 3′-OH termini were used
as donor substrates, cyclization of the donor and byproducts
from repeated ligation of the donor with already formed ligation
products occurred significantly (Figure 3b,c and supporting
information; 28, 33-35). The latter could only be avoided when
the 5′-phosphate oligoribonucleotide and the partner strand to
be ligated formed a highly stable duplex prior to the treatment
with ligase and when the ligation position was situated in a
loop or bulge region of the resulting product (Figure 3b; 28).
Among donors, 5′-terminal pyrimidines are known to be
slightly preferred over purines. Among acceptors, 3′-terminal
adenosines are the best acceptors, cytidines and guanosines show
intermediate reactivity, and terminal uridine residues are poor
substrates.20 With respect to ligation site 4 of our target
sequence, optimal requirements are therefore fulfilled (Figure
3d-e; Table 2). We also found that 2′-Se-methyl nucleosides in
the 5′-terminal position of the donor (14) can be ligated as
efficiently as standard nucleosides (Figure 3d and supporting
information; 39). T4 RNA ligase recognizes only the first
nucleobase and first two phosphates of the donor. With respect
to the acceptor substrates the requirements are more stringent.
The recognition site for the acceptor encompasses three nucleo-
sides and their bridging phosphates.20 Thus, a 2′-Se-methyl
nucleoside in the third position from the 3′-OH terminus did
hamper ligation almost completely compared to the unmodified
counterpart (Figure 3c and Supporting Information; 12 versus
29), whereas a 2′-Se-methyl nucleoside positioned four nucleo-
sides apart from the 3′-OH terminus did not (Figure 3c; 13
versus 31). Remarkably, the modified nucleoside N2-methylgua-
nosine in the second position from the 3′-OH terminus of the
acceptor 42 did also not reduce ligation rate or yield (Figure
3e; Table 2, 45, 46). Figure 4 illustrates two typical sets of
HPLC traces for the ligation of sequences comprising the
naturally occurring methylations, one referring to the 2′-Se-
methyluridine target sequence 45, the other referring to the 2′-
Se-methylcytidine target 46.
The identities of the ligation products without selenium were
confirmed by co-injection with the identical sequences obtained
by solid-phase synthesis (28, 34, 38, 44; supporting information).
For the subset of ligations listed in Table 2 we have isolated
the product compounds by anion exchange HPLC and charac-
terization was done by MALDI-TOF mass spectrometry.
Figure 3. (a) Target stem-loop motif of rat spliceosomal U6 snRNA.
Arrows indicate ligation sites. (b-e) Conception of the different ligation
experiments with T4 RNA ligase. Details are given in the text. USe, 2′-Se-
methyluridine; CSe, 2′-Se-methylcytidine.
The set of experiments presented above has shown that the
selenium-modified RNAs are stable throughout enzymatic
ligation conditions. Our U6 snRNA target sequences are only
about 30 nucleotides in length. We have chosen this length to
enable a broad study with respect to the optimization of various
ligation parameters and thereby permitting a fast and reliable
characterization of the full-length products. The ligation ap-
proach is amenable for longer selenium-containing RNAs and
also for the isolation of larger amounts. This is currently in
progress in our laboratory for different target sequences of larger
size and with multiple Se labels.
nucleotides 57-78 should be stabilized by additional cytidine-
guanosine base pairs and should contain all the naturally
occurring nucleoside modifications beside the selenium label
(Figure 3). As indicated by arrows, we considered four potential
sites for the enzymatic ligation of such an oligoribonucleotide
(Figure 3a). First experience was collected from the ligation of
(20) Arn, E. A.; Abelson, J. In RNA Structure and Function; Simons, R. W.;
Grunberg-Manago, M., Eds.; CSHL Press: New York, 1998; pp 695-
726.
(21) Massenet, S.; Mougin, A.; Branlant, C. In Modification and Editing of RNA;
Grosjean, H., Benne, R., Eds.; ASM Press: Washington, 1998, pp 201-
227.
9
J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004 1145

A R T I C L E S
Ho¨bartner and Micura
Table 2. Selected Ligation Experiments of an U6 snRNA Stem-Loop Motif with T4 RNA Ligasea
isolated yield
molecular weight
c
260 nm
no
ligation producta
scale nmol
ligation conditionsb
calcd yield, %
OD
nmol
%
calcd amu
foundd amu
38
5′-GCGUGGCCCCUGCGCAAGG
AUGACACGCUp-3′
20
100 µM, 0.20 U/µL, 37 °C, 4 h
48
2.3
7.5
37
9392.7
9399.7
40
41
44
45
46
5′-GCGUGGCCCCUGCGCAAGG
AUGACACGCUSep-3′
5′-GCGUGGCCCCUGCGCAAGG
AUGACACSeGCUp-3′
5′-GCGUGGCmCCmCmUGCGCA
AmGm2GAUGACmACGCUp-3′
5′-GCGUGGCmCCmCmUGCGCA
AmGm2GAUGACmACGCUSep-3′
5′-GCGUGGCmCCmCmUGCGCA
AmGm2GAUGACmACSeGCUp-3′
20
8
120 µM, 0.30 U/µL, 22 °C, 12 h
40 µM, 0.21 U/µL, 37 °C, 4 h
80 µM, 0.20 U/µL, 37 °C, 4 h
100 µM, 0.30 U/µL, 22 °C, 15 h
140 µM, 0.40 U/µL, 22 °C, 12 h
52
44
45
60
44
2.7
1.0
1.1
2.0
2.2
8.8
3.3
3.5
6.4
7.1
44
41
35
49
41
9469.6
9469.6
9476.8
9553.8
9553.8
9475.8
9474.6
9482.8
9559.9
9561.8
10
13
17
a USe, 2′-Se-methyluridine; CSe, 2′-Se-methylcytidine; Cm, 2′-O-methylcytidine; Am, 2′-O-methyladenosine; m2G, N2-methylguanosine. b Concentrations
of each ligation fragment and of T4 RNA ligase in ligation buffer (50 mM Tris.HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP), reaction temperature,
and reaction time. c Yield calculated from HPLC traces taking into account different extinction coefficients of fragments and ligation products. d MALDI-
TOF MS; molecular weights detected as [M + H]+.
Figure 4. (a) Enzymatic ligation of 2′-Se-methyluridine and 2′-Se-methylcytidine derivatized oligoribonucleotides with T4 RNA ligase (c(42,17,18) ) 120 µM
each; 0.40 U/µL ligase). HPLC traces of reaction mixtures at ligation start (b), after 12 h at 22 °C (c), and of the isolated products (d). Anion exchange
HPLC: Dionex DNAPac (4 × 250 mm), 80 °C, 1 mL/min, 0-60% B in 45 min; A, 25 mM Tris·HCl, 6 M urea, pH 8.0; B, same as A + 0.5 M NaClO4;
see also Table 2.
Conclusion
(DTT) during the deprotection procedure. Moreover, various
Se-labeled sequences of an U6 snRNA stem-loop motif of
about 30 nucleotides in size have been achieved by enzymatic
ligation with T4 RNA ligase. This basic ligation study involving
the chemically synthesized Se-labeled oligoribonucleotides
should demonstrate the flexibility of the selenium approach and
the potential for an extension to even larger target sequences.
This may attract the interest of structural biologists dealing with
RNA X-ray crystallography.
With the present work we have documented a robust protocol
for the synthesis of 2′-Se-methyluridine and -cytidine phos-
phoramidites and their incorporation into oligoribonucleotides.
By solid-phase synthesis sequences with up to 25 nucleotides
and including up to two Se labels are readily accessible. Key
feature for the successful and reproducible preparation is the
use of millimolar amounts of threo-1,4-dimercapto-2,3-butandiol
9
1146 J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004

Selenium-Modified Oligoribonucleotides
A R T I C L E S
required till gas bubbles started to occur in the yellow mixture. The
solution was stirred at room temperature for 1 h and the almost colorless
solution was injected into a solution of 2 (500 mg; 0.945 mmol) in dry
THF (10 mL). The reaction mixture was stirred at room temperature
for 2 h and monitored by TLC (CH2Cl2/MeOH ) 92/8). When the
reaction was complete, water (2 mL) was added and the pH of the
mixture was adjusted to 7 by the dropwise addition of 20% acetic acid.
The solvents were evaporated under reduced pressure at 40 °C, water
was added, and the product was repeatedly extracted into ethyl acetate.
The combined ethyl acetate extracts were washed with saturated sodium
chloride solution. The organic layer was dried over Na2SO4, and the
solvent was evaporated. The crude product was purified by column
chromatography on SiO2 (CH2Cl2 + 1-2% MeOH). Yield: 540 mg
of 3 as colorless foam (92%). TLC (CH2Cl2/MeOH 92/8): Rf 0.75. 1H
NMR (300 MHz, CDCl3): δ 2.12 (s, 3 H, SeCH3); 2.62 (d, J ) 3.0
Hz, 1 H, HO-C(3′)); 3.51-3.61 (m, 3 H, H2-C(5′), H-C(2′)); 3.81
(s, 6 H, 2 OCH3); 4.19 (m, 1 H, H-C(4′)); 4.39 (m, 1 H, H-C(3′));
5.38 (d, J ) 8.3 Hz, 1 H, H-C(5)); 6.18 (d, J ) 7.5 Hz, 1 H, H-C(1′));
6.85 (m, 4 H, H-C(ar)); 7.25-7.38 (m, 9 H, H-C(ar)); 7.78 (d, J )
8.3 Hz, 1 H, H-C(6)); 8.11 (s, br, 1 H, NH) ppm. FT ICR ESI-MS
m/z calcd for C31H32N2O7Se [M + Na]+ 647.127 56; found 647.127 56.
Experimental Section
Materials. 1H, 13C, and 31P NMR spectra were recorded on a Bruker
DRX 300 MHz or Varian Unity 500 MHz instrument. The chemical
shifts are reported relative to TMS and referenced to the residual proton
signal of the deuterated solvents: CDCl3 (7.26 ppm) or DMSO-d6 (2.49
ppm) for 1H NMR spectra; CDCl3 (77.0 ppm) or DMSO-d6 (39.5 ppm)
for 13C NMR spectra. 31P-shifts are relative to external 85% phosphoric
acid. UV spectra were recorded on a Varian Cary 100. Analytical thin-
layer chromatography (TLC) was carried out on silica 60F-254 plates.
Compounds on the plates were visualized by dipping into a solution
of ethanol (180 mL), anisaldehyde (10 mL), concentrated H2SO4 (10
mL), and acetic acid (2 mL) and subsequent heating with a heat gun.
Flash column chromatography was carried out with silica gel 60 (230-
400 mesh). Packing of silica gel columns was performed with 1% Et3N
added to the corresponding starting eluent. All reactions were carried
out under Ar atmosphere. Chemical reagents and solvents were
purchased from commercial suppliers and used without further purifica-
tion. Organic solvents for reactions were dried overnight over freshly
activated molecular sieves (4 Å).
Phosphoramidite Synthesis. We note that selenium is highly toxic
and that handling of the selenium-derivatized compounds requires
wearing of protective clothing and a good fume hood.
5′-O-(4,4′-Dimethoxytrityl)-2′-deoxy-2′-Se-methyluridine 3′-(2-Cy-
anoethyl)diisopropylphosphoramidite (4) was prepared with slight
modifications according to ref 5. Compound 3 (300 mg, 0.48 mmol)
was dissolved in a mixture of ethyldimethylamine (520 µL; 4.8 mmol)
in dry dichloromethane (5 mL) under argon. After 15 min at room
temperature, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (170
mg; 0.72 mmol) was slowly added and the solution was stirred at room
temperature for 2 h. The reaction was quenched by the addition of
MeOH (0.2 mL). The reaction mixture was diluted with dichloro-
methane, extracted with saturated sodium bicarbonate solution, and dried
over Na2SO4, and the solvent was evaporated. The crude product was
purified by column chromatography on SiO2 (ethyl acetate/hexane 1/1
to 7/3 (+1% NEt3)) and isolated as a 1:1 mixture of diastereoisomers.
Yield: 368 mg of 4 as colorless foam (93%). TLC (ethyl acetate/hexane
2,2′-O-Anhydro(â-D-arabinofuranosyl)uracil (1) was prepared with
slight modifications according to ref 16. To a stirred suspension of
diphenyl carbonate (1.92 g; 9.0 mmol) in N,N-dimethylformamide (2.2
mL), uridine (2.0 g; 8.2 mmol) was added. The slurry was heated to
80 °C at which point sodium bicarbonate (25 mg; 0.3 mmol) was added
and the reaction mixture was heated to 120 °C. Gas evolution was
observable, a clear solution resulted which soon deposited the solid
product. Once the evolution of gas subsided (after 3-4 h), the
suspension was cooled to room temperature, the precipitate was isolated
by filtration, and the product was washed with methanol. Yield: 1.4 g
1
of 1 as white powder (75%). TLC (CH2Cl2/MeOH 85/15): Rf 0.2. H
NMR (300 MHz, DMSO-d6): δ 3.17-3.28 (m, 2 H, H2-C(5′)); 4.06
(m, 1 H, H-C(4′)); 4.37 (m, 1 H, H-C(3′)); 4.95 (triplettoid, 1 H,
HO-C(5′)); 5.18 (d, J ) 6.2 Hz, 1 H, H-C(2′)); 5.84 (m, 2 H, H-C(5)
+ HO-C(3′)); 6.29 (d, J ) 6.2 Hz, 1 H, H-C(1′)); 7.82 (d, J ) 7.4
Hz, 1 H, H-C(6)) ppm.
1
7/3): Rf 0.6. H NMR (500 MHz, CDCl3): δ 1.05-1.28 (m, 24 H,
((CH3)2CH)2N); 2.04, 2.08 (2s, 6 H, SeCH3); 2.37, 2.62 (2m, 4 H, CH2-
CN); 3.41-3.69 (m, 10 H, ((CH3)2CH)2N, POCH2, H2-C(5′)); 3.79
(2s, 12 H, OCH3); 3.90, 3.93 (2m, 4 H, POCH2); 4.24, 4.30 (2m, 2 H,
H-C(4′)); 4.63, 4.68 (2m, 2 H, H-C(3′)); 5.25, 5.31 (2d, 2 H, J )
8.0 Hz, H-C(5)); 6.33 (d, 2 H, H-C(1′)); 6.84 (m, 8 H, H-C(ar));
7.24-7.37 (m, 18 H, H-C(ar)); 7.80 (d, 2 H, J ) 8.0 Hz, H-C(6));
8.36 (s, br, 2 H, NH) ppm. 31P NMR (121 MHz, CDCl3): 151.14, 151.88.
FT ICR ESI-MS m/z calcd for C40H49N4O8PSe [M + Na]+ 847.235 60,
found 847.232 85; calcd [M + K]+ 863.209 54, found 863.208 37.
2,2′-O-Anhydro-5′-O-(4,4′-dimethoxytrityl)(â-D-arabinofurano-
syl)uracil (2) was prepared with slight modifications according to ref
16. To a stirred suspension of 1 (500 mg; 2.2 mmol) in dry pyridine (5
mL) at room temperature, 4,4′-dimethoxytrityl chloride (820 mg, 2.4
mmol) was added in two portions over a period of 2 h. The suspension
turned into a clear solution during 1-3 h. The resulting orange solution
was stirred for another 4-6 h and the reaction was monitored by TLC
(CH2Cl2/MeOH ) 9/1); when the reaction was complete, methanol (0.5
mL) was added and the solution was stirred for 30 min. The solvents
were evaporated; the residue was dissolved in dichloromethane and
extracted with 5% citric acid, water, and sodium bicarbonate solution.
The organic layer was dried over Na2SO4 and evaporated. The crude
product was purified by column chromatography on SiO2 (CH2Cl2/
MeOH ) 98/2 to 90/10 (+1% NEt3)). Yield: 775 mg of 2 as colorless
3′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-
2′-Se-methyluridine (6). Compound 3 (500 mg, 0.80 mmol) was
dissolved in DMF (4 mL) and mixed with imidazole (218 mg; 3.20
mmol) and tert-butyldimethylsilyl chloride (242 mg; 1.60 mmol). The
colorless solution was kept under argon and stirred at room temperature
for 18 h. The solvent was evaporated under reduced pressure. The
product was precipitated from the resulting oil by the addition of water,
isolated by filtration, washed with water and hexanes, and dried under
high vacuum. The crude product was used without further purification.
An analytical sample was prepared by column chromatography on silica
gel (CH2Cl2 + 0-3% MeOH). Yield: 560 mg of 6 as white solid
(95%). TLC (CH2Cl2/MeOH 98/2): Rf 0.65. UV (MeOH): λ(ꢀ) 260
1
foam (67%). TLC (CH2Cl2/MeOH 9/1): Rf 0.6. H NMR (500 MHz,
DMSO-d6): δ 2.80 (dd, J ) 10.0, 7.5 Hz, 1 H, H-C(5′)); 2.93 (dd, J
) 10.0, 4.0 Hz, 1 H, H-C(5′)); 3.72 (s, 6 H, 2 OCH3); 4.20 (m, 1 H,
H-C(4′)); 4.29 (m, 1 H, H-C(3′)); 5.19 (d, J ) 6.0 Hz, 1 H, H-C(2′));
5.86 (d, J ) 7.5 Hz, 1 H, H-C(5)); 5.94 (s, br, 1 H, HO-C(3′)); 6.31
(d, J ) 6.0 Hz, 1 H, H-C(1′)); 6.82 (m, 4 H, H-C(ar)); 7.12-7.28
(m, 9 H, H-C(ar)); 7.93 (d, J ) 7.5 Hz, 1 H, H-C(6)) ppm.
5′-O-(4,4′-Dimethoxytrityl)-2′-deoxy-2′-Se-methyluridine (3) was
prepared with slight modifications according to ref 5. Sodium boro-
hydride (215 mg, 5.67 mmol) was placed in a sealed 25-mL two-necked
round-bottom flask, dried on high vacuum for 15 min to deplete oxygen,
kept under argon, and suspended in dry THF (7 mL). Dimethyl
diselenide (180 µL; 1.89 mmol) was slowly injected to this suspension,
followed by dropwise addition of anhydrous ethanol; 0.5 mL were
1
(11 600) nm (L mol-1 cm-1). H NMR (300 MHz, CDCl3): δ -0.02
(s, 3 H, SiCH3); 0.12 (s, 3 H, SiCH3); 0.86 (s, 9 H, SiC(CH3)3); 2.04
(s, 3 H, SeCH3); 3.38 (dd, J ) 11.0, 2.3 Hz, 1 H, H1-C(5′)); 3.47 (m,
1 H, H-C(2′)); 3.56 (dd, J ) 11.0, 3.0 Hz, 1 H, H2-C(5′)); 3.81 (s,
6 H, 2 OCH3)); 4.09 (m, 1 H, H-C(4′)); 4.48 (m, 1 H, H-C(3′)); 5.33
(d, J ) 8.3 Hz, 1 H, H-C(5)); 6.34 (d, J ) 7.1 Hz, 1 H, H-C(1′));
6.85 (d, J ) 8.7 Hz, 4 H, H-C(ar)); 7.25-7.38 (m, 9H, H-C(ar));
7.88 (d, J ) 8.3 Hz, 1 H, H-C(6)); 7.95 (s, br, 1 H, NH) ppm. 13C
NMR (75 MHz, CDCl3): δ -4.80 (SiCH3); -4.73 (SiCH3); 3.35
9
J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004 1147

A R T I C L E S
Ho¨bartner and Micura
(SeCH3); 18.07 (SiC(CH3)3); 25.66 (SiC(CH3)3); 48.61 (C(2′)); 55.24
(OCH3); 62.38 (C(5′)); 73.50 (C(3′)); 85.57 (C(4′)); 87.27; 90.01 (C(1′));
102.50 (C(5)); 113.28 (C(ar)); 127.24, 127.97, 128.18, 130.12 (C(ar));
134.94, 135.08; 140.18 (C(6)); 144.05, 150.31, 158.80, 163.12 ppm.
FT ICR ESI-MS m/z calcd for C37H46N2O7SeSi [M + Na]+ 761.214 21,
found 761.214 96.
isolated by column chromatography on SiO2 (CH2Cl2/MeOH ) 98/2
to 94/6). Yield: 150 mg of 10 as colorless foam (87%). TLC (CH2-
Cl2/MeOH 97/3): Rf 0.40. UV (MeOH): λ(ꢀ) 272 (7200), 300 (7400)
1
nm (L mol-1 cm-1). H NMR (300 MHz, CDCl3): δ 2.17 (s, 3 H,
SeCH3); 2.19 (s, 3 H, COCH3); 3.00 (s, br, 1 H, HO-C(3′)); 3.54 (2dd,
J ) 3.0, 11.3 Hz, 2 H, H2-C(5′)); 3.66 (m, 1 H, H-C(2′)); 3.81 (s, 6
H, 2 OCH3)); 4.15 (m, 1 H, H-C(4′)); 4.39 (m, 1 H, H-C(3′)); 6.30
(d, J ) 4.5 Hz, 1 H, H-C(1′)); 6.86 (d, J ) 9.2 Hz, 4 H, H-C(ar));
7.14 (d, J ) 7.5 Hz, 1 H, H-C(5)); 7.26-7.42 (m, 9 H, H-C(ar));
8.30 (d, J ) 7.5 Hz, 1 H, H-C(6)); 9.48 (s, br, 1 H, NH) ppm. 13C
NMR (75 MHz, CDCl3): δ 4.65 (SeCH3); 24.75 (COCH3); 51.46
(C(2′)); 55.23 (OCH3); 62.25 (C(5′)); 70.01 (C(3′)); 84.43 (C(4′)); 87.20;
90.22 (C(1′)); 97.00 (C(5)); 113.37 (C(ar)); 127.17, 128.03, 128.15,
130.06 (C(ar)); 135.21, 135.42, 144.23; 144.49 (C(6)); 155.35, 158.76,
162.88, 170.80 ppm. FT ICR ESI-MS m/z calcd for C33H35N3O7Se [M
+ H]+ 666.172 17, found 666.172 08, calcd [M + Na]+ 688.154 14;
found 688.153 44.
N4-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-Se-methylcyti-
dine 3′-(2-Cyanoethyl)diisopropylphosphoramidite (11). Compound
10 (110 mg; 0.165 mmol) was dissolved in a mixture of ethyldimethy-
lamine (180 µL, 1.6 mmol) in dry dichloromethane (5 mL) under argon.
After 15 min at room temperature, 2-cyanoethyl N,N-diisopropylchlo-
rophosphoramidite (60 mg; 0.25 mmol) was slowly added and the
solution was stirred at room temperature for 2 h. The reaction was
quenched by the addition of methanol (0.2 mL). The reaction mixture
was diluted with dichloromethane, extracted with saturated sodium
bicarbonate solution, and dried over Na2SO4, and the solvent was
evaporated. The crude product was purified by column chromatography
on SiO2 (ethyl acetate/hexane 7/3 (+1% NEt3)) and isolated as a 1:1
mixture of diastereoisomers. Yield: 118 mg of 11 as colorless foam
(82%). TLC (CH2Cl2/ MeOH 96/4): Rf 0.55. UV (MeOH): λ(ꢀ) )
274 (7830), 300 (7950) nm (L mol-1 cm-1). 1H NMR (500 MHz,
CDCl3): δ 0.98-1.25 (m, 24 H, ((CH3)2CH)2N); 2.11 (s, 6 H, COCH3);
2.15, 2.17 (2s, 6 H, SeCH3); 2.33, 2.59 (2m, 4 H, CH2CN); 3.41-3.70
(m, 12 H, ((CH3)2CH)2N, POCH2, H2-C(5′), H-C(2′)); 3.78 (2s, 12
H, OCH3); 3.80-3.93 (m, 2 H, POCH2); 4.29 (m, 2 H, H-C(4′)); 4.55,
4.61 (2m, 2 H, H-C(3′)); 6.36 (m, 2 H, H-C(1′)); 6.81 (m, 8 H,
H-C(ar)); 6.89, 6.95 (2d, br, 2 H, H-C(5)); 7.22-7.37 (m, 18 H,
H-C(ar)); 8.30 (m, 2 H, H-C(6)); 8.78 (s, br, 2 H, NH) ppm. 31P
NMR (121 MHz, CDCl3) δ 151.23, 151.49 ppm. FT ICR ESI-MS m/z
calcd for C42H52N5O8PSe [M + H]+ 866.280 22, found 866.278 66.
3′-O-tert-Butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-
2′-Se-methylcytidine (8). A solution of 6 (500 mg; 0.68 mmol) and
DMAP (10 mg; 0.08 mmol) in dry dichloromethane (5 mL) was treated
under argon with triethylamine (950 µL; 6.8 mmol). Subsequently,
2,4,6-triisopropylbenzenesulfonyl chloride (310 mg; 1.02 mmol) was
slowly added. The solution was stirred for 1 h at room temperature
The reaction mixture was diluted with dichloromethane, extracted with
saturated sodium bicarbonate solution, dried over Na2SO4, and evapo-
rated. The intermediate 7 (yellow foam) was dried under vacuum for
0.5 h, then dissolved in THF (10 mL), treated with aqueous ammonia
(15 mL; 32%), and stirred at room temperature overnight. The solvents
were evaporated and the crude product was purified by column
chromatography on SiO2 (CH2Cl2/MeOH ) 98/2 to 92/8). Yield: 395
mg of 8 as slightly yellow foam (79%). TLC (CH2Cl2/MeOH 95/5):
Rf 0.45. UV (MeOH): λ(ꢀ) 260 (10 300) nm (L mol-1 cm-1). 1H NMR
(300 MHz, CDCl3): δ -0.08 (s, 3 H, SiCH3); 0.08 (s, 3 H, SiCH3);
0.81 (s, 9 H, SiC(CH3)3); 2.13 (s, 3 H, SeCH3); 3.30 (dd, J ) 2.3, 10.5
Hz, 1 H, H1-C(5′)); 3.58 (m, 1 H, H-C(2′)); 3.63 (dd, J ) 3.0, 10.5
Hz, 1 H, H2-C(5′)); 3.80 (s, 6 H, 2 OCH3)); 4.14 (m, 1 H, H-C(4′));
4.46 (m, 1 H, H-C(3′)); 5.30 (d, J ) 7.5 Hz, 1 H, H-C(5)); 6.38 (d,
J ) 3.9 Hz, 1 H, H-C(1′)); 6.84 (d, J ) 7.6 Hz, 4 H, H-C(ar)); 7.26-
7.40 (m, 9 H, H-C(ar)); 8.12 (d, J ) 7.5 Hz, 1 H, H-C(6)) ppm. 13
C
NMR (75 MHz, CDCl3): δ -4.90 (SiCH3); -4.69 (SiCH3); 3.08
(SeCH3); 18.07 (SiC(CH3)3); 25.68 (SiC(CH3)3); 49.12 (C(2′)); 55.27
(OCH3); 61.95 (C(5′)); 72.10 (C(3′)); 84.65 (C(4′)); 87.00; 90.71 (C(1′));
94.90 (C(5)); 113.24, 113.27 (C(ar)); 127.13, 127.92, 128.34, 130.25
(C(ar)); 135.27, 135.35; 141.23 (C(6)); 144.27, 158.78, 165.45 ppm.
FT ICR ESI-MS m/z calcd for C37H47N3O6SeSi [M + Na]+ 760.230 18,
found 760.228 80.
N4-Acetyl-3′-O-tert-butyldimethylsilyl-5′-O-(4,4′-dimethoxytrityl)-
2′-deoxy-2′-Se-methylcytidine (9). A solution of 8 (200 mg; 0.27
mmol) in dry pyridine (0.5 mL) was cooled to 0 °C under argon, treated
with acetic anhydride (65 µL; 0.68 mmol), allowed to warm to room
temperature, and stirred for 30 min. The reaction was quenched by the
addition of methanol (0.3 mL). The solvents were evaporated; the oily
residue was dissolved in dichloromethane and extracted with 5% citric
acid, water, and saturated sodium bicarbonate solution. The organic
layer was dried over Na2SO4 and evaporated. The crude product was
purified by column chromatography on SiO2 (CH2Cl2/MeOH ) 99/1
to 95/5). Yield: 197 mg of 9 as colorless foam (94%). TLC (CH2Cl2/
MeOH 97/3): Rf 0.65. UV (MeOH): λ(ꢀ) 272 (6000), 300 (5800) nm
(L mol-1 cm-1). 1H NMR (300 MHz, CDCl3): δ -0.06 (s, 3 H, SiCH3);
0.08 (s, 3 H, SiCH3); 0.82 (s, 9 H, SiC(CH3)3); 2.15 (s, 3 H, SeCH3);
2.27 (s, 3 H, COCH3); 3.38 (dd, J ) 2, 10.5 Hz, 1 H, H1-C(5′)); 3.58
(m, 1 H, H-C(2′)); 3.67 (dd, J ) 2, 10.5 Hz, 1 H, H2-C(5′)); 3.82 (s,
6 H, 2 OCH3)); 4.20 (m, 1 H, H-C(4′)); 4.44 (m, 1 H, H-C(3′)); 6.39
(d, J ) 3.9 Hz, 1 H, H-C(1′)); 6.86 (d, J ) 8.0 Hz, 4 H, H-C(ar));
7.08 (d, J ) 7.5 Hz, 1 H, H-C(5)); 7.28-7.40 (m, 9 H, H-C(ar));
8.47 (d, J ) 7.5 Hz, 1 H, H-C(6)); 10.37 (s, br, 1 H, NH) ppm. 13C
NMR (75 MHz, CDCl3): δ -5.00 (SiCH3); -4.74 (SiCH3); 3.18
(SeCH3); 18.03 (SiC(CH3)3); 24.78 (COCH3); 25.62 (SiC(CH3)3); 49.26
(C(2′)); 55.22 (OCH3); 61.55 (C(5′)); 71.68 (C(3′)); 85.04 (C(4′)); 87.24;
91.15 (C(1′)); 96.99 (C(5)); 113.30 (C(ar)); 127.26, 127.96, 128.34,
130.19 (C(ar)); 135.02, 135.13, 143.88; 144.69 (C(6)); 155.06, 158.83,
163.11, 170.97 ppm. FT ICR ESI-MS m/z calcd for C39H49N3O7SeSi
[M + Na]+ 802.240 80, found 802.241 21.
Oligoribonucleotide Synthesis. 5′-O-DMT-2′-O-TOM-protected
nucleoside phosphoramidites (A, C, G, U), the corresponding 2′-O-
TOM-standard nucleoside-CPG supports (500 Å), phosphoramidites of
N4-acetyl-5′-O-DMT-2′-O-methylcytidine, N6-benzoyl-5′-O-DMT-2′-
O-methyladenosine, N6-Pac-5′-O-DMT-2′-deoxyadenosine, and N2-iPr-
Pac-5′-O-DMT-2′-deoxyguanosine were obtained from GlenResearch,
Sterling, VA, or Xeragon AG, Switzerland. The phosphoramidite of
5′-O-DMT-N2-methyl-O6-(4-nitrophenyl-2-ethyl)-2′-O-TOM-guano-
sine was synthesized as described in ref 22. Anthracene hexaethylene
glycol phosphoramidite was synthesized as described in ref 23. CPG
support 47 for the synthesis of 3′-phosphate oligoribonucleotides was
prepared according to ref 24; phosphoramidite 48 for 5′-phosphate
labeling was prepared according to ref 25 (Chart 1). 2′-Se-methyluridine
CPG support 49 was prepared via compound 5.
5′-O-(4,4′-Dimethoxytrityl)-2′-deoxy-2′-Se-methyluridine 3′-(4-
nitrophenyl hexandioate) (5). Compound 3 (165 mg; 0.26 mmol) was
dissolved in pyridine (2 mL) and DMF (2 mL). The mixture was treated
with DMAP (33 mg; 0.26 mmol) and bis-(4-nitrophenyl)hexandioate
(475 mg; 1.23 mmol). The reaction solution was stirred at room
temperature for 24 h. The solvents were evaporated the residue dissolved
N4-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-Se-methylcyti-
dine (10). Compound 9 (200 mg; 0.26 mmol) was treated with 0.5 mL
of 1 M TBAF/0.5 M AcOH in THF. The solution was stirred at room
temperature for 2.5 h. The solvent was evaporated and the product was
(22) Ho¨bartner, C.; Kreutz, C.; Flecker, E.; Ottenschla¨ger, E.; Pils, W.;
Grubmayr, K.; Micura R. Monatsh. 2003, 134, 851-873.
(23) Stuhlmann, F.; Ja¨schke, A. J. Am. Chem. Soc. 2002, 124, 3238-3244.
(24) Garc´ıa-Echeverr´ıa, C. Tetrahedron Lett. 1997, 38, 8933-8934.
(25) Horn, T.; Urdea, M. S. Tetrahedron Lett. 1986, 27, 4705-4708.
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1148 J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004

Selenium-Modified Oligoribonucleotides
A R T I C L E S
Chart 1. Solid-Phase Supports and Phosphorylation Reagent
dryness. Removal of the 2′-O-silyl ethers was achieved by treatment
with TBAF‚3H2O in THF (1 M, 0.90 mL) for at least 12 h at room
temperature. The reaction was quenched by the addition of Tris·HCl
(1 M, pH 7.4, 0.95 mL). The volume of the solution was reduced to 1
mL and directly applied on a Sephadex G 10 column (30 × 1.5 cm)
controlled by UV detection at 270 nm. The product was eluted with
water and evaporated to dryness.
All oligoribonucleotides were purified by anion-exchange chroma-
tography on a semipreparative Dionex DNAPac column (9 × 250 mm)
at 80 °C. Flow rate, 2 mL/min; eluant A, 25 mM Tris·HCl, 6 M urea,
in H2O (pH 8.0); eluant B, 25 mM Tris·HCl, 0.5 M NaOCl4, 6 M urea,
in H2O (pH 8.0); detection at 265 nm, ∆5-10% B in A within 20 min.
Fractions containing the purified oligonucleotide were desalted by
loading onto a C18 SepPak cartridge (Waters/Millipore), followed by
elution with 0.1-0.2 M (Et3NH)HCO3, water, and then H2O/CH3CN
(6/4). Combined fractions containing the oligonucleotide were lyoph-
ilized to dryness.
Enzymatic Ligations Using T4 RNA Ligase. General procedure
for the ligations of 28, 33-35, and 39: 2 nmol containing aliquots
from aqueous stock solutions of the oligonucleotides to be ligated were
mixed, 5 µL of ligation buffer (500 mM Tris‚HCl, 100 mM MgCl2,
100 mM DTT, 10 mM ATP, pH 7.8 at 25 °C) were added and the
volume was adjusted to 50 µL by the addition of water to give a final
oligonucleotide concentration of 40 µM for each oligonucleotide
fragment and a final ligation buffer concentration of 50 mM Tris‚HCl,
10 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 7.8 at 25 °C. The reaction
solution was heated to 90 °C for 4 min and allowed to cool to 25 °C
with a cooling rate of 1 °C/min. Then, 0.5 µL (10 U) of T4 RNA ligase
in storage solution (New England Biolabs, 20000 U/mL in 50 mM
KCl, 10 mM Tris‚HCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol)
were added to give a final T4 RNA ligase concentration of 0.2 U/µL.
The reaction solution was incubated at 37 °C. After 4 h aliquots (5
µL) of the reaction solution were inactivated by heat (95 °C; 3 min)
and analyzed by anion-exchange HPLC (Dionex DNAPac column, 4
× 250 mm, 80 °C, 0-60% B in 45 min; A, 25 mM Tris·HCl, 6 M
urea, pH 8.0; B, 25 mM Tris·HCl, 6 M urea, 0.5 M sodium perchlorate,
pH 8.0) and denaturating polyacrylamide gel electrophoresis (15%
polyacrylamide, 7 M urea). Ligations of 38, 40, 41, and 44-46: ligation
experiments were performed as described above; scale, 5-20 nmol;
concentration of each ligation fragment, 40-140 µM; concentration
of T4 RNA ligase, 0.2-0.4 U/µL; ligation temperature, reaction time,
37 °C, 4 h or 22 °C, 12-15 h (see Table 2). The T4 RNA ligase was
heat inactivated at 95 °C for 3 min and the ligation product isolated by
anion-exchange HPLC (Dionex DNAPac column, 4 × 250 mm) as
described for synthetic products in the preceding section. The identities
of the isolated ligation products were confirmed by MALDI-TOF mass
spectrometry (Table 2).
in dichloromethane, extracted with half-saturated sodium bicarbonate
solution, and dried over Na2SO4, and the solvent was evaporated. The
crude product was purified by column chromatography on SiO2
(CH2Cl2/acetone 8/1 to 7/1). Yield: 195 mg of 4 as colorless foam
(85%). TLC (CH2Cl2/acetone 7/1): Rf 0.7. 1H NMR (300 MHz,
CDCl3): δ 1.79-1.83 (m, 4 H, 2 CH2); 2.09 (s, 3 H, SeCH3); 2.47 (m,
2 H, CH2); 2.66 (m, 2 H, CH2); 3.54 (2 dd, J ) 2.5, 8.9 Hz, 2 H,
H2-C(5′)); 3.64 (dd, J ) 6.3, 9.0 Hz, 1 H, H-C(2′)); 3.80 (s, 6 H, 2
OCH3); 4.18 (m, 1 H, H-C(4′)); 5.34 (d, J ) 9.0 Hz, 1 H, H-C(5));
5.53 (dd, J ) 2.7, 6.3 Hz, 1 H, H-C(3′)); 6.32 (d, J ) 9.0 Hz, 1 H,
H-C(1′)); 6.85 (m, 4 H, H-C(ar)); 7.25-7.34 (m, 11 H, H-C(ar));
7.76 (d, J ) 9.0 Hz, 1 H, H-C(6)); 8.27 (d, J ) 10.0 Hz, 2 H, H-C(ar))
ppm. 13C NMR (75 MHz, CDCl3): δ 4.92 (SeCH3); 24.01 (COCH3);
33.57 (CH2); 33.84 (CH2); 45.21 (C(2′)); 55.26 (OCH3); 63.05 (C(5′));
74.98 (C(3′)); 83.29 (C(4′)); 87.58; 88.67 (C(1′)); 102.89 (C(5)); 113.36
(C(ar)); 122.40; 125.20; 127.36; 128.09; 130.06, 130.13 (C(ar)); 134.75;
134.90; 139.82 (C(6)); 144.02; 145.29; 150.46, 155.32; 158.83; 163.03;
170.72; 172.22 ppm.
Preparation of Solid Support 49. To a solution of the active ester
5 (120 mg, 0.14 mmol) in 2 mL of DMF was added long-chain
alkylamino 500 Å CPG (0.5 g) followed by ethyldiisopropylamine (200
µL, 1.17 mmol) and pyridine (20 µL, 0.25 mmol). The mixture was
agitated for 24 h at room temperature. After filtration, the solids were
washed with DMF, methanol, and dichloromethane, dried, suspended
in a mixture of 5 mL capping solution A/B (1/1; see below), and shaken
for 20 min at room temperature. After filtration, the solids were washed
with DMF, methanol, and dichloromethane and dried under high
vacuum. Loading of the support was 35 µmol/g.
All oligoribonucleotides were synthesized on a Pharmacia Gene
Assembler Plus following DNA/RNA standard methods: detritylation,
dichloroacetic acid/1,2-dichloroethane (4/96, 2 min); coupling, phos-
phoramidites/acetonitrile (0.1 M × 120 µL, 2.5 min); activation by
benzyl thiotetrazole/acetonitrile (0.35 M × 360 µL); capping, A, Ac2O/
sym-collidine/acetonitrile (20/30/50), B, DMAP/acetonitrile (0.5 M),
A/B ) 1/1 (1 min); alternatively, when support 47 was used, capping,
A, Ac2O/sym-collidine/THF (1/1/8), B, N-methylimidazole in THF (16/
84); oxidation, I2 (10 mM) in acetonitrile/sym-collidine/H2O (10/1/5),
(1 min). Amidite solutions, tetrazole solutions, and acetonitrile were
dried over activated molecular sieves overnight. All sequences were
synthesized trityl-off.
Oligoribonucleotide Deprotection and Purification. Deprotection
of the selenium-containing oligoribonucleotides and cleavage from the
solid support were achieved with MeNH2 in EtOH (8 M, 0.75 mL)
and MeNH2 in water (40%, 0.75 mL) containing 10-150 mM DTT
for 5-6 h at room temperature; the solution was then evaporated to
Acknowledgment. Funding from the Austrian Science Fund
is greatly acknowledged. Harald Mittendorfer (Innsbruck) is
acknowledged for synthetic contributions, Dr. Kathrin Breuker
(Innsbruck; Ithaca, NY) for recording FT-ICR mass spectra. We
thank Professor Dinshaw Patel (MSKCC, NY) and his group
for crystallization screenings and enthusiasm on the project.
Professor Scott Strobel and Mary Stahley (Yale, CT) are thanked
for successful crystallization involving sequence 24, Andre`s
Ja¨schke and Sonja Keiper (Heidelberg) for advice in the syn-
thesis of anthracene hexaethyleneglycol phosphoramidite.
Supporting Information Available: HPLC traces concerning
the deprotection optimization of Se-labeled oligoribonucleotides
(12, 23, and 24); HPLC traces and PAGE of selected ligation
experiments with T4 RNA ligase; trityl assays for strand assem-
bly; MALDI-TOF and FT-ICR-MS spectra (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA038481K
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J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004 1149