Synthesis and incorporation of the phosphoramidite derivative of 2′- O-photocaged 3′-S-thioguanosine into oligoribonucleotides: Substrate for probing the mechanism of RNA catalysis

Article abstract of DOI:10.1021/jo4028374

Oligoribonucleotides containing 3′-S-phosphorothiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. Photocaged 3′-S- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite starting from N²-isobutyrylguanosine in nine steps with 10.2% overall yield. Oligonucleotides containing the 2′-O-(o- nitrobenzyl)-3′-S-guanosine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction.

Full text of DOI:10.1021/jo4028374

Note
pubs.acs.org/joc
Synthesis and Incorporation of the Phosphoramidite Derivative of
2′‑O‑Photocaged 3′‑S‑Thioguanosine into Oligoribonucleotides:
Substrate for Probing the Mechanism of RNA Catalysis
Nan-Sheng Li,† Nicole Tuttle,‡ Jonathan P. Staley,§ and Joseph A. Piccirilli*,†,‡
‡
§
†Department of Biochemistry & Molecular Biology, Department of Chemistry, and Department of Molecular Genetics & Cell
Biology, University of Chicago, Chicago, Illinois 60637, United States
S
* Supporting Information
ABSTRACT: Oligoribonucleotides containing 3′-S-phosphorothiolate linkages possess properties that can reveal deep
mechanistic insights into ribozyme-catalyzed reactions. “Photocaged” 3′-S- RNAs could provide a strategy to stall reactions at the
chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the
synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite starting from N2-isobutyrylguanosine in nine steps with
10.2% overall yield. Oligonucleotides containing the 2′-O-(o-nitrobenzyl)-3′-S-guanosine nucleotide were then constructed,
characterized, and used in a nuclear pre-mRNA splicing reaction.
ligonucleotides containing phosphorothiolate linkages, in
monitor the chemical step. Despite broad utility of these
substrates in the investigation of phosphoryl transfer reactions,
complex assembly processes and conformational changes that
accompany biological catalysis frequently mask analysis of the
chemical step. Photocaging provides a well-established strategy
to protect functional groups from chemical reactions until
released by UV irradiation.9−11 In this respect, having access to
“photocaged” 3′-S-modified RNAs could provide a strategy to
stall reactions at the chemical stage and release them only after
assembly steps have occurred. Moreover, photocaging provides
the added advantage of increasing the stability of these
phosphorothiolate-modified oligonucleotides by eliminating
side reactions involving 2′-O-transphosphorylation.1
O
which sulfur replaces the 3′- or 5′-bridging oxygen
connected to the ribofuranose ring (Figure 1, II and III), serve
The 2′-O-(o-nitrobenzyl) derivatives of uridine,12 adeno-
sine,13 cytidine,13 and guanosine14 have been synthesized and
applied to the synthesis of oligoribonucleotides containing one
or multiple 2′-O-photoliable groups (Figure 1, I*).12,13,15−18
Dinucleotides and trinucleotides containing a 2′-O-(o-nitro-
benzyl) group were usually synthesized via a solution method
involving condensation between the 3′-phosphate or 5′-
phosphate of one nucleoside and the 5′-OH or 3′-OH group
of another nucleoside.12,13,17 Longer oligoribonucleotides (>3-
mer) containing 2′-O-(o-nitrobenzyl) groups could be
Figure 1. Structures of wild type, 3′-S-modified, and 5′-S-modified
oligonucleotides.
as powerful biochemical probes to investigate fundamental
features of enzyme catalysis.1−4 RNAs containing a 3′-S-
phosphorothiolate linkage have been used in metal ion rescue
experiments to elucidate the mechanism of metalloribozymes
such as the group I intron, the group II intron, and the
spliceosome.5−8 These studies rely on careful comparison of
the 3′-O and 3′-S substrates and must include assays that
Received: December 20, 2013
Published: March 17, 2014
© 2014 American Chemical Society
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Note
Scheme 1
efficiently synthesized via solid-phase synthesis using 3′-
phosphoramidites15,16 or 3′-H-phosphonates of 2′-O-(o-nitro-
benzyl) nucleosides.18 After removal of the 2′-O-(o-nitro-
benzyl) group by photolysis, these RNAs could initiate efficient
and accurate ribozyme-catalyzed reactions.15,16 This RNA-
caging approach has also been used to investigate spliceosome
assembly during pre-mRNA splicing through introducing a 2′-
O-(o-nitrobenzyl) group into the branch adenosine nucleotide,
which blocks the first step of splicing, thereby allowing the
study of the precatalytic stages during assembly.19
The synthesis of oligonucleotides containing a 2′-O-photo-
liable group and 3′-S-phosphorothiolate linkage (Figure 1, II*)
has not yet been reported. Here we report the synthesis of the
phosphoramidite derivative of the 2′-O-(o-nitrobenzyl)-3′-
thioguanosine and its application for the construction of
oligonucleotides containing a 2′-O-photocaged 3′-S-phosphoro-
thiolate linkage via solid-phase synthesis.
For the synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine
phosphoramidite 8 (Scheme 1), we adapted previous
approaches for the synthesis of 2′-O-TBS-3′-thioguanosine
phosphoramidite20 and 2′-O-methyl-3′-thioguanosine phos-
phoramidite.21 We chose the o-nitrobenzyl group in this
study because the starting material o-nitrobenzyl bromide is
commercially available and relatively inexpensive. Additionally,
use of the o-nitrobenzyl group is well-established for the
synthesis of 2′-O-photocaged RNAs. N2-Isobutyryl-2′-O-(o-
nitrobenzyl)guanosine (2) was prepared from N2-isobutyryl-
guanosine (1) in 47% yield as described previously, except that
we quenched the reaction with dilute aqueous HCl.22 Reaction
of 2 with (tert-butyl)diphenylsilyl chloride gave the correspond-
ing 5′-silyl derivative in 94% yield. In the presence of 1.4 molar
equiv of DMAP, the 5′-silyl derivative reacted with 1.05 molar
equiv of trifluoromethanesulfonyl chloride at 0 °C to give the
corresponding 3′-triflate derivative 3 in 62% yield. When 2.0
molar equiv of DMAP and 1.5 molar equiv of trifluoro-
methanesulfonyl chloride were used, the reaction afforded 3′-
triflate derivative 3 in 50% yield along with the guanosine 3′,O6-
ditriflate derivative (∼13% yield). Subsequent SN2 substitution
with 4 molar equiv of NaBr in refluxing acetone afforded 3′-β-
bromo derivative 4 in 95% yield. Reaction of 4 with KSAc in
DMF at 60 °C gave a mixture of the 3′-S-acetyl and 3′,4′-
unsaturated derivatives in a 1.3/1 ratio. Desilylation of the
mixture with TBAF·xH2O/AcOH in THF and purification by
silica gel chromatography gave the desired pure 5′-deprotected
derivative 5 in 54% yield over the two steps. Although the 3′-β-
iodo derivative could be prepared by SN2 substitution of 3 with
NaI in 96% yield, the subsequent reaction with KSAc, followed
by desilylation of the reaction mixture, produced predominantly
the 3′,4′-unsaturated derivative and gave the desired 5′-
deprotected derivative 5 only in 19% yield. Protection of 5
with DMTCl in pyridine for 24 h afforded the 5′-O-DMT ether
6 in 95% yield. The 3′-S-acetyl group of 6 was selectively
removed by treatment with a 5:1 mixture of guanidine
hydrochloride and guanidine23 to give 3′-SH derivative 7 in
90% yield. Phosphitylation of compound 7 with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite yielded the corre-
sponding 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphorami-
dite 8 in 85% yield.
Incorporation of phosphoramidite 8 into an 18-mer
oligonucleotide was then accomplished by manual coupling
using our previously described protocol.24 The modified
oligonucleotide, 5′-UUU AG3′S,2′‑O‑(o‑NBn) A GGU UGC UGC
UUU-3′ (2′-O-photocaged-3′-S-RNA), was obtained in 1.5%
yield after standard deprotection and reverse-phase HPLC
purification. The presence of the modification was confirmed
by MALDI-TOF MS (Figure S1 in Supporting Information).
Additionally, the oligonucleotide was 5′-radiolabeled with γ32-
ATP and further characterized by UV deprotection, alkaline
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Figure 2. Alkaline hydrolysis, silver ion cleavage, and RNase T1 cleavage of 2′-photocaged 3′-O-substrate (5′-UUU AG3′O,2′‑O‑(o‑NBn) A GGU UGC
UGC UUU-3′) and 2′-photocaged 3′-S-substrate (5′-UUU AG3′S,2′‑O‑(o‑NBn) A GGU UGC UGC UUU-3′): (A) 5′-pUUUAG2′‑OH,3′‑SH-3′, (B) 5′-
pUUUAG2′‑O,3′‑SP-3′ (2′-O,3′-S-cyclic phosphoryl 5-mer), (C) 5′-pUUUAG2′‑OH,3′‑SP-3′ (3′-S-phosphoryl 5-mer), and (D) 5′-pUUUA-
G2′‑O‑(o‑NBn),3′‑SH-3′. The numbers in the picture are assigned for the ladders of alkaline hydrolysis and T1 treatment of these two 18-mer RNAs.
Figure 3. (a) Schematic for synthesis of ACT1 yeast splicing substrates. RNA is shown as black lines and boxes; the DNA splint is shown in gray.
The boxes represent the exons, and the black line represents the intron. The red star indicates the position of the radiolabel. The modified 2′-O-
photocaged residue is the last nucleotide of the intron. (b) Schematic of the two steps of splicing. (c) Exon ligation of the 3′-O-pc and 3′-S-pc
substrates in an in vitro splicing assay (60 mM K2PO4 (pH 7), 3% PEG 8000, 2.5 mM MgCl2, 2 mM ATP, 40% yeast extract in the initial
incubation); “metal” indicates the addition of 0.5 mM MgCl2, MnCl2, or nothing as indicated in the main text.
hydrolysis, silver ion cleavage, and RNase T1 cleavage (Figure
2). For comparison, we prepared the corresponding 2′-
photocaged wild-type RNA, 5′-UUU AG3′O,2′‑O‑(o‑NBn) A GGU
UGC UGC UUU-3′ (2′-O-photocaged-3′-O-RNA), by solid-
phase synthesis using 2′-O-(o-nitrobenzyl)guanosine phos-
phoramidite. The UV-deprotected 3′-S-RNA was readily
cleaved under alkaline conditions (pH 9) to form predom-
inantly three 5-nucleotide products. Three cleavage products
from top to bottom on line 8 of Figure 2 were assigned as (A)
5′-pUUUAG2′‑OH,3′‑SH-3′, (B) 5′-pUUUAG2′‑O,3′‑SP-3′ (2′-O,3′-
catalyzed strand scission than the 2′-photocaged 3′-S-oligo
(Figure 2, compare lane 7 to 8). Exposure to silver ion of both
2′-O-photocaged 3′-S-RNA and UV-deprotected 3′-S-RNA
resulted in the cleavage products assigned as 5′-pUUUA-
G
2′‑O‑(o‑NBn),3′‑SH-3′ and 5′-pUUUAG2′−OH,3′‑SH-3′, respectively
(Figure 2, lanes 11 and 12).26 These results confirm that the
RNA contains a 3′-S-phosphorothiolate linkage, and the ratio of
the D and A product band indicates that UV irradiation
converted ∼90% of the 2′-O-photocaged 3′-S-RNA to 3′-S-
RNA (lane 12). Moreover, neither 2′-O-photocaged guanosine
3′-O-RNA nor 2′-O-photocaged guanosine 3′-S-RNA was
cleaved by RNase T1 at the photocaged residue until after
UV deprotection (Figure 2, compare lanes 13 to 14 and 15 to
16). These results confirm that the RNA contains a 2′-
photocaged 3′-phosphorothiolate linkage at the designated
S-cyclic phosphosphate 5-mer), and (C) 5′-pUUUAG2′‑OH,3′‑SP
-
3′ (3′-S-phosphate 5-mer).25,26 These product assignments
correspond to those identified for alkaline cleavage of the
dinucleotide pIspU.26 As anticipated, the UV-deprotected 3′-S-
oligonucleotide was more susceptible toward hydroxide-
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Note
g, 7.30 mmol) was added, and the mixture was stirred at rt for 5 h. The
reaction was neutralized with 1 N HCl. The mixture was evaporated,
and the residue was purified by silica gel chromatography, eluting with
4−6% methanol in dichloromethane to give the product 2a14,22 as a
yellow foam (1.12 g, 47% yield).
position and shows that the photocage can be removed
efficiently.
We next tested whether the 2′-O-photocaged 3′-S-oligo-
nucleotide could be used to stage nuclear pre-mRNA splicing
reactions at the chemical step. Nuclear pre-mRNA splicing is
catalyzed by the spliceosome, a complex ribonucleoprotein
particle that undergoes assembly steps and rearrangements en
route to the chemical steps.27 We incorporated the photocaged
3′-S-oligonucleotide into a widely used ACT1 yeast splicing
substrate and used it in metal rescue experiments with the
spliceosome (Figure 3a).28 The oligonucleotide was designed
to contain the photocaged residue at the intron terminus, such
that the phosphorothiolate was in the leaving group position for
the second step of splicing. To assemble the full-length
substrate, we 32P-radiolabeled the O- and S-containing
oligonucleotides and then ligated them to two RNA oligo-
nucleotides generated by in vitro transcription (Figure 3a).
Next we incubated these RNA substrates in splicing-competent
Saccharomyces cerevisiae extract so that spliceosomes could
assemble on them and catalyze branching (Figure 3b). The
lariat intermediate product of the branching step runs
aberrantly slowly and can be seen as the top band of the
splicing gel (Figure 3c).
With the photocage in place, the spliceosomes stalled prior
to exon ligation regardless of the identity of the atom (O or S)
in the leaving group position (Figure 3c, lanes 1 and 7). To
remove the photocage and permit exon ligation (Figure 3b), we
irradiated spliceosomes for 5 min with 365 nm UV light (lanes
4−6 and 10−12). To initiate exon ligation, we then added
additional MgCl2, MnCl2, or nothing and assayed for exon
ligation. As expected, appearance of the product of exon
ligation of the 3′-O-pc substrate proceeded efficiently in the
UV-treated spliceosomes (lanes 4−6) but not without
irradiation (lanes 1−3). Exon ligation of the 3′-S-pc substrate
only proceeded efficiently with UV treatment and in the
presence of MnCl2 (compare lane 12 with lanes 7−11). These
results demonstrate that the photocage comprises an effective
block to splicing chemistry and that, upon removal, splicing
occurs in the presence of an added metal ion. The dependence
of the 3′-S-pc substrate reaction on the “thiophilic” manganese-
(II) ion reflects the role of the metal ion in leaving group
stabilization.8
5′-O-(tert-Butyldiphenylsilyl)-N2-isobutyryl-2′-O-(o-nitro-
benzyl)-3′-O-trifluoromethylsulfonylguanosine (3). To a stirred
solution of N2-isobutyryl-2′-O-(o-nitrobenzyl)guanosine (2) (2.312 g,
4.73 mmol) in dry pyridine (20 mL) was added tert-butyldiphenylsilyl
chloride (1.82 mL, 7.10 mmol) under argon. The mixture was stirred
at rt for 24 h, then quenched with CH3OH (4.0 mL), and evaporated
to a syrup. The residue was dissolved in CH2Cl2 and washed with
H2O. The organic layer was dried over anhydrous MgSO4. After
filtration and removal of solvent, the residue was isolated by silica gel
chromatography, eluting with 2% CH3OH in CH2Cl2 to give 5′-O-
(tert-butyldiphenylsilyl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)guanosine
as a white foam: 3.24 g (94% yield); 1H NMR (CDCl3/TMS) δ 12.20
(brs, 1H), 9.95 (brs, 1H), 7.97 (s, 1H), 7.96 (d, 1H, J = 8.8 Hz), 7.70−
7.60 (m, 5H), 7.53 (m, 1H), 7.45−7.25 (m, 7H), 6.03 (d, 1H, J = 2.8
Hz), 5.28 (d, 1H, J = 15.0 Hz), 5.14 (d, 1H, J = 15.0 Hz), 4.61 (m,
1H), 4.28 (m, 1H), 4.22 (m, 1H), 4.05 (m, 1H), 3.88 (m, 1H), 2.84
(m, 1H), 1.26 (d, 3H, J = 4.8 Hz), 1.24 (d, 3H, J = 4.8 Hz), 1.02 (s,
9H); 13C NMR (CDCl3) δ 179.6, 155.6, 147.9, 147.7, 146.9, 136.8,
135.5, 135.4, 134.0, 133.8, 132.7, 132.4, 129.84, 129.80, 128.7, 128.4,
127.8, 127.7, 124.6, 121.2, 86.9, 84.6, 83.3, 69.1, 68.8, 63.0, 36.1, 26.8,
19.0, 18.9, 18.8; HRMS calcd for C37H43N6O8Si [MH+] 727.2906,
found 727.2903. Under argon, to a solution of 5′-O-(tert-butyldi-
phenylsilyl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)guanosine (3.24 g, 4.46
mmol) and DMAP (763 mg, 6.25 mmol) in dry CH2Cl2 (30 mL) at 0
°C was added CF3SO2Cl (0.50 mL, 4.7 mmol). After the mixture was
stirred at 0 °C for 3 h, the reaction was quenched with ice water (20
mL) and the mixture stirred for 15 min. The organic layer was
separated, and the aqueous layer was extracted with CH2Cl2 (2 × 30
mL). The organic layers were combined, washed with brine, and
subsequently dried over anhydrous MgSO4. After filtration and
removal of solvent, the residue was purified by silica gel
chromatography, eluting with 1.5−2% methanol in CH2Cl2 to give 3
1
as a white foam: 2.37 g (62% yield); H NMR (CDCl3/TMS + a few
drops of D2O) δ 8.00 (d, 1H, J = 8.4 Hz), 7.85 (s, 1H), 7.70−7.50 (m,
6H), 7.50−7.30 (m, 7H), 6.03 (d, 1H, J = 4.8 Hz), 5.71 (m, 1H), 5.30
(d, 1H, J = 14.8 Hz), 5.09 (d, 1H, J = 14.8 Hz), 4.89 (t, 1H, J = 4.8
Hz), 4.46 (m, 1H), 4.08 (dd, 1H, J = 3.4, 12.0 Hz), 3.86 (dd, 1H, J =
3.2, 12.0 Hz), 2.63 (m, 1H), 1.27 (d, 3H, J = 6.0 Hz), 1.26 (d, 3H, J =
6.0 Hz), 1.04 (s, 9H); 13C NMR (CDCl3) δ 178.8, 155.4, 147.8, 147.7,
147.0, 137.1, 135.6, 135.5, 134.3, 133.3, 132.2, 132.0, 130.5, 130.4,
128.8, 128.6, 128.18, 128.15, 124.8, 122.1, 118.6, 86.4, 82.2, 81.9, 80.1,
69.9, 61.9, 36.6, 26.9, 19.2, 19.02, 18.98; HRMS calcd for
C38H42N6O10F3SiS [MH+] 859.2399, found 859.2399.
In summary, we have developed an efficient synthesis of the
phosphoramidite derivative of 2′-O-(o-nitrobenzyl)-3′-thio-
guanosine starting from N2-isobutyrylguanosine in nine steps.
From this phosphoramidite, an 18-nucleotide RNA containing
a 2′-O-photoliable group adjacent to a 3′-S-phosphorothiolate
linkage was successfully synthesized by the solid-phase
synthesis. Irradiation with UV light released the photocaging
group without affecting the integrity of the 3′-S modification.
This capability can enable effective use of the 3′-sulfur
modification to perform metal rescue experiments even in
systems that undergo complex assembly and conformational
changes en route to the chemical step. As proof-of-principle, we
have incorporated this modified RNA into a model yeast pre-
mRNA splicing substrate and used it to probe for catalytic
metal ion interactions in the spliceosome.8
3′-β-Bromo-5′-O-(tert-butyldiphenylsilyl)-N2-isobutyryl-2′-
O-(o-nitrobenzyl)guanosine (4). A mixture of 3 (1.641 g, 1.91
mmol) and NaBr (786 mg, 7.64 mmol) in acetone (20 mL) was
heated under reflux for 23 h under argon. The solvent was removed,
and the residue was isolated by silica gel chromatography, eluting with
2% methanol in CH2Cl2 to give 4 as pale yellow foam: 1.43 g (95%
1
yield); H NMR (CDCl3/TMS) δ 12.15 (brs, 1H), 9.76 (brs, 1H),
8.07 (d, 1H, J = 7.2 Hz), 7.84 (s, 1H), 7.80−7.60 (m, 6H), 7.55−7.30
(m, 7H), 6.00 (s, 1H), 5.56 (d, 1H, J = 14.8 Hz), 5.11 (d, 1H, J = 14.8
Hz), 4.60−4.47 (m, 3H), 4.11 (dd, 1H, J = 5.8, 10.6 Hz), 4.03 (dd,
1H, J = 5.8, 10.6 Hz), 2.83 (m, 1H), 1.27 (d, 6H, J = 7.2 Hz), 1.09 (s,
9H); 13C NMR (CDCl3) δ 179.5, 155.6, 148.0, 147.4, 146.9, 136.7,
135.59, 135.57, 134.3, 133.6, 132.8, 130.0, 128.8, 128.7, 127.89,
127.87, 124.9, 121.4, 90.6, 89.5, 82.4, 69.3, 64.8, 50.3, 36.3, 26.8, 19.2,
19.1, 18.9 ppm; HRMS calcd for C37H42N6O7SiBr [MH+] 789.2062,
found 789.2059.
3′-S-Acetyl-N2-isobutyryl-2′-O-(o-nitrobenzyl)-3′-thioguano-
sine (5). Under argon to a solution of 4 (2.99 g, 3.78 mmol) in dry
DMF (25 mL) was added potassium thioacetate (1.30 g, 11.4 mmol),
and the mixture was stirred at 60 °C for 7 h. After the solvent was
removed under reduced pressure, the residue was partitioned between
a saturated aqueous NaHCO3 solution/brine (v/v, 1:1) and CH2Cl2.
EXPERIMENTAL SECTION
■
N2-Isobutyryl-2′-O-(o-nitrobenzyl)guanosine (2). Under
argon, N2-isobutyrylguanosine 1 (1.72 g, 4.86 mmol) was treated
with NaH (307 mg, 95%, 12.15 mmol) in DMF (40 mL) at 0 °C. After
hydrogen gas generation ceased (45 min), o-nitrobenzyl bromide (1.58
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The organic layer was dried over MgSO4. After filtration and removal
of the solvent, the residue was dissolved in THF (45 mL), and AcOH
(1.23 mL, 21.7 mmol) and n-Bu4NF·3H2O (2.74 g, 8.69 mmol) were
added. The mixture was stirred at rt for 14 h, then diluted with
CH2Cl2, washed with saturated aqueous NaHCO3 and brine, and dried
over MgSO4. After filtration and removal of solvent, the residue was
purified by silica gel chromatography, eluting with 3% MeOH in
was removed, and the residue was purified by silica gel
chromatography, eluting with 0−5% acetone in CH2Cl2 containing
0.5% Et3N to give product as a white foam: 0.146 g (85% yield); 31P
NMR (CD3CN)
δ 164.6, 159.2 ppm; HRMS calcd for
C51H60N8O10PS [MH+] 1007.3885, found 1007.3884.
5′-O-(Dimethoxytrityl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)-
guanosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite
(9). To a solution of 5′-O-(dimethoxytrityl)-N2-isobutyryl-2′-O-(o-
nitrobenzyl)guanosine22 (47 mg, 0.059 mmol) in dry CH2Cl2 (5.0
mL) under Ar were added N,N-diisopropylethylamine (52 μL, 0.30
mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (42 mg,
0.18 mmol), and 1-methylimidazole (5.0 μL, 0.059 mmol). The
mixture was stirred at rt until all starting material was consumed (1 h).
The reaction was quenched with MeOH (1 mL) and stirred for 5 min.
After the solvent was removed, the crude product was purified by silica
gel chromatography, eluting with 1% MeOH in CH2Cl2 containing
0.5% Et3N, to give the corresponding phosphoramidite as a yellow
foam: 56 mg (95% yield); 31P NMR (CD3CN) δ 152.8, 152.6; HRMS
calcd for C51H60N8O11P [MH+] 991.4119, found 991.4110.
1
CH2Cl2 to give 5 as a white foam: 1.12 g (54% yield); H NMR
(CD3OD/TMS) δ 8.37 (s, 1H), 8.00 (dd, 1H, J = 1.2, 8.4 Hz), 7.71
(d, 1H, J = 6.8 Hz), 7.66 (dt, 1H, J = 1.2, 7.6 Hz), 7.51 (dt, 1H, J = 1.2,
7.8 Hz), 6.19 (d, 1H, J = 2.0 Hz), 5.25 (d, 1H, J = 13.6 Hz), 5.03 (d,
1H, J = 13.6 Hz), 4.55−4.44 (m, 2H), 4.25−4.18 (m, 1H), 3.92 (dd,
1H, J = 2.4, 12.8 Hz), 3.72 (dd, 1H, J = 2.8, 12.8 Hz), 2.75 (m, 1H),
2.36 (s, 3H), 1.25 (d, 3H, J = 4.4 Hz), 1.23 (d, 3H, J = 4.4 Hz); 13C
NMR (CDCl3) δ 196.0, 181.6, 157.4, 149.7, 149.1, 139.2, 134.7, 134.4,
130.8, 130.0, 125.8, 121.5, 89.2, 86.4, 85.2, 70.8, 61.4, 44.6, 37.0, 30.4,
19.4 19.3 ppm; HRMS calcd for C23H27N6O8S [MH+] 547.1606,
found 547.1606.
3′-S-Acetyl-5′-O-(dimethoxytrityl)-N2-isobutyryl-2′-O-(o-
nitrobenzyl)-3′-thioguanosine (6). To a solution of 5 (861 mg,
1.58 mmol) in dry pyridine (15 mL) under argon was added DMTCl
(1.07 g, 3.15 mmol). The mixture was stirred at rt for 24 h and
quenched with MeOH. The solvent was removed, the residue was
partitioned between 5% aqueous NaHCO3 and CH2Cl2. The organic
layer was washed with brine and dried over MgSO4. After filtration and
removal of solvent, the residue was isolated by silica gel
chromatography, eluting with 0−1% MeOH in CH2Cl2 containing
Oligonucleotide Synthesis. The oligonucletide 5′-UUU
AG3′S,2′‑O‑oNBnA GGU UGC UGC UUU-3′ (ACT1-3'-S) was
synthesized on an Expedite 8900 DNA synthesizer by manual
coupling of phosphoramidite 8 through a modified protocol as
previously described.24 Following the standard oligonucleotide
deprotections29 (a, concentrated NH4OH/EtOH, 3:1 (v/v), 55 °C,
17 h; b, TEA-3HF/TEA/NMP, 65 °C, 1.5 h), the oligonucleotide
(1.5% yield) was obtained by reverse-phase HPLC purification (C18
column, 0−30% aceonitrile/100−70% 0.1 M TEAA pH 7.0 over 30
min) and confirmed by the MALDI-TOF MS: calcd for (M + NH4)
5854.8, found 5853.1.
Oligonucleotide Characterization. Alkaline hydrolysis: 4k cpm
of the 5′-radiolabeled oligonucleotide (1 μL) with or without UV
deprotection (UVP-B1000, 365 nm, 4 min) was treated with NaHCO3
(pH 9, 50 mM, 2 μL) in a total volume of 10 μL solution at 90 °C for
10 min. Formamide loading dye (2×, 10 μL) was added, and the
mixture was run on a 20% dPAGE gel.
Silver ion cleavage: 4k cpm of the 5′-radiolabeled oligonucleotides
(2 μL) with or without UV deprotection (UVP-B1000, 365 nm, 4
min) was treated with AgNO3 (100 mM, 0.4 μL) in a total volume of
20 μL solution in the dark at rt for 60 min. DTT (100 mM, 0.6 μL)
was then added, and the mixture was spun at full speed for 5 min. A 15
μL aliquot of solution was withdrawn, added to 2× formamide loading
dye (15 μL), and run on a 20% dPAGE gel.
Rnase T1 treatment: 4k cpm of the 5′-radiolabeled oligonucleotides
(1 μL) with or without UV deprotection (UVP-B1000, 365 nm, 4
min) was combined with 8 M urea (pH 9, 6 μL), 200 mM sodium
citrate (pH 5, 1 μL), and 1 unit of RNase T1 in a final reaction volume
of 9 μL. After a 10 min incubation at 37 °C, 2× formamide loading dye
(8 μL) was added and the sample was run on a 20% dPAGE gel.
Synthesis of ACT1 Yeast Splicing Substrates.8 The ACT1-1-
373 (nucleotides 1−373) was synthesized by in vitro transcription
from a plasmid template linearized with HindIII restriction and
containing ACT1-1-373 followed by an HDV ribozyme sequence. In
cases where HDV cleavage was inefficient during transcription, the
RNA was resuspended in Tris (10 mM, pH 7.5) and MgCl2 (20 mM).
Ribozyme cleavage was induced via 2−4 cycles of 90 °C for 1 min, rt
for 15 min, and 37 °C for 15 min. The buffer conditions were then
adjusted for T4 PNK treatment of the transcript to remove the 2′,3′-
cyclic phosphate left by the ribozyme. The ACT1-392-590
(nucleotides 392−590) was synthesized by in vitro transcription
using a PCR-derived template generated using plasmid bJPS149. As
the subsequent ligation requires a 5′-monophosphate group, a 4-fold
excess of GMP over GTP was included in the transcription reaction.
ACT1 ligation reactions consisted of 500 pmol of ACT1-1-373, 50
pmol of oligonucleotide ACT1-3′-O or ACT1-3′-S, and 500 pmol of
ACT1-392-590. The RNA was hybridized to 50 pmol of ACT1 splint
in buffer TEN50 (10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 50 mM
NaCl) on a thermal cycle by heating to 90 °C for 2 min followed by
reduction of the temperature by 1 °C for 1 min to 24 °C, then cooling
to 4 °C for 5 min. T4 DNA ligase (∼100 pmol, synthesized in-house)
1
0.5% Et3N to give 6 as a pale yellow foam: 1.28 g (95% yield); H
NMR (CDCl3/TMS) δ 12.04 (brs, 1H), 9.18 (brs, 1H), 8.08 (d, 1H, J
= 8.0 Hz), 7.98 (s, 1H), 7.82 (d, 1H, J = 7.6 Hz), 7.69 (m, 1H), 7.50−
7.15 (m, 10H), 6.80 (d, 4H, J = 8.8 Hz), 6.09 (s, 1H), 5.51 (d, 1H, J =
15.6 Hz), 5.15 (d, 1H, J = 15.2 Hz), 4.84 (dd, 1H, J = 5.2, 11.2 Hz),
4.42 (d, 1H, J = 4.8 Hz), 4.35 (m, 1H), 3.77 (s, 6H), 3.51 (dd, 1H, J =
2.8, 11.2 Hz), 3.32 (m, 1H), 2.71 (m, 1H), 2.30 (s, 3H), 1.26 (d, 3H, J
= 7.2 Hz), 1.24 (d, 3H, J = 6.9 Hz); 13C NMR (CDCl3) δ 193.9, 179.0,
158.6, 155.5, 147.7, 147.1, 146.8, 144.3, 137.0, 135.6, 135.5, 134.34,
134.25, 130.1, 128.6, 128.5, 128.2, 127.9, 127.0, 124.8, 122.0, 113.1,
88.7, 86.5, 84.8, 82.9, 69.5, 61.4, 55.3, 43.7, 36.5, 30.5, 19.0 18.8 ppm;
HRMS calcd for C44H45N6O10S [MH+] 849.2912, found 849.2922.
5′-O-(Dimethoxytrityl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)-
3′-thioguanosine (7). To a solution of guanidine hydrochloride (108
mg, 1.13 mmol) in a mixed solvent of MeOH/CH2Cl2 (9:1, v/v, 10
mL) was added NaOMe (0.5 M in MeOH, 0.40 mL, 0.20 mmol) at 0
°C. After the mixture was stirred at 0 °C for 10 min, the solution was
transferred to the flask containing 6 (160 mg, 0.188 mmol) in dry
CH2Cl2 (10 mL). The mixture was warmed to rt and stirred for 3 h,
and then partitioned between H2O and CH2Cl2. The organic layer was
separated, washed with brine, and dried over anhydrous MgSO4. The
solvent was removed, and the residue was isolated by silica gel
chromatography, eluting with 1−2% MeOH in CH2Cl2 containing
0.5% Et3N to give the product as a white foam: 137 mg (90% yield);
1H NMR (CDCl3/TMS) δ 12.08 (brs, 1H), 9.20 (brs, 1H), 8.07 (d,
1H, J = 7.2 Hz), 8.06 (s, 1H), 7.89 (d, 1H, J = 7.6 Hz), 7.68 (m, 1H),
7.53−7.18 (m, 10H), 6.83 (d, 4H, J = 7.6 Hz), 6.07 (s, 1H), 5.67 (d,
1H, J = 15.6 Hz), 5.21 (d, 1H, J = 15.6 Hz), 4.20 (m, 1H), 4.15 (d, 1H,
J = 5.2 Hz), 3.77 (s, 6H), 3.70 (m, 1H), 3.62 (m, 1H), 3.49 (dd, 1H, J
= 2.8, 11.2 Hz), 2.75 (m, 1H), 1.26 (d, 6H, J = 7.0 Hz); 13C NMR
(CDCl3) δ 179.2, 158.7, 155.5, 147.9, 147.1, 146.7, 144.3, 136.1, 135.5,
134.6, 130.15, 130.06, 128.5, 128.4, 128.14, 128.08, 127.1, 124.8,
121.9, 113.4, 88.1, 86.7, 86.5, 85.6, 69.1, 60.6, 55.3, 38.6, 36.4, 19.1,
18.9 ppm; HRMS calcd for C42H43N6O9S [MH+] 807.2807, found
807.2819.
5′-O-(Dimethoxytrityl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)-
3′-thioguanosine-3′-S-(cyanoethyl N,N-diisopropylphos-
phoramidite) (8). Under argon to a solution of 7 (137 mg, 0.17
mmol) in dry dichloromethane (10 mL) were added N,N-diisopropyl-
ethylamine (0.15 mL, 0.86 mmol), 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (96 μL, 0.43 mmol), and 1-methylimidazole
(6.2 μL, 0.078 mmol). The mixture was stirred at rt for 1 h, quenched
with MeOH (1 mL), and stirred for an additional 5 min. The solvent
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dx.doi.org/10.1021/jo4028374 | J. Org. Chem. 2014, 79, 3647−3652

The Journal of Organic Chemistry
Note
or T4 RNA ligase 2 (2 units, New England Biolabs) was then added,
and reactions were incubated at 37 °C for 4 h. The ligation reactions
were DNase-treated (RNase-free DNase) for 15 min to remove splint,
phenol−chloroform extracted, and ethanol precipitated before
purification on 6% denaturing polyacrylamide gel. Bands containing
full-length ACT1 pre-mRNA were excised and recovered by passive
elution in TEN250 buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA,
250 mM NaCl) overnight at 4 °C. Yields typically ranged from 200 to
600 fmol, enough for 50−150 splicing reactions.
Splicing Reactions. Yeast splicing-competent extracts were
prepared using the liquid nitrogen method, as described in the
literature.30 In vitro splicing reactions consisted of 32P-labeled
substrates (0.2−0.4 nM), 40% yeast extract that was pretreated with
1 mM EDTA, 3% PEG 8000, 60 mM K2PO4 (pH 7), 3.5 mM MgCl2,
and 2 mM ATP. Reactions were incubated at 20 °C for 20 min, then
on ice for 5 min for 365 nm UV light treatment. Metal ion
concentrations were adjusted to 4 mM with either MgCl2 or MnCl2,
and the reactions were incubated at 20 °C for another 20 min. The
reactions were then quenched and analyzed by 6% dPAGE gel as
described previously.8
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
MALDI-TOF MS of 2′-photocaged 3′-S-RNA, H NMR and
13C NMR spectra of compounds 3−7, 1H NMR and 31P NMR
of phosphoramidites 8 and 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
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220.
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Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S.; Usman, N.
Nucleic Acids Res. 1995, 23, 2677−2684.
AUTHOR INFORMATION
■
(30) Umen, J. G.; Guthrie, C. Genes Dev. 1995, 9, 855−968.
Corresponding Author
*E-mail: jpicciri@uchicago.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Hao Huang, Dr. Sandip Shelke, and Dr.
Sebastian M. Fica for helpful discussions and critical comments
on the manuscript. This work was supported by an NIH grant
(R01GM088656) to J.A.P. and J.P.S.
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