Synthesis and biochemical application of 2′-O-methyl-3′-thioguanosine as a probe to explore group I intron catalysis

Article abstract of DOI:10.1016/j.bmc.2008.03.061

Oligonucleotides containing 3′-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here, we have synthesized 2′-O-methyl-3′-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme reaction. We developed an efficient method for the synthesis of 2′-O-methyl-3′-thioguanosine phosphoramidite in eight steps starting from 2′-O-methyl-N²-(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-phase synthesis, we used this new analogue to investigate whether the 3′-oxygen of the guanosine cofactor in the Tetrahymena ribozyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 2′-hydroxyl group. We show that regardless of whether the guanosine cofactor bears a 3′-oxygen or 3′-sulfur leaving group, replacing the adjacent 2′-hydroxyl group with a 2′-methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen bond donated by the guanosine 2′-hydroxyl group contributes to catalytic function in a manner distinct from the U_-1 2′-hydroxyl group.

Full text of DOI:10.1016/j.bmc.2008.03.061

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5754–5760
Synthesis and biochemical application of
20-O-methyl-30-thioguanosine as a probe to explore
group I intron catalysis
Jun Lu, Nan-Sheng Li, Raghuvir N. Sengupta and Joseph A. Piccirilli*
Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology, and Chemistry,
The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA
Received 8 February 2008; accepted 24 March 2008
Available online 27 March 2008
Abstract—Oligonucleotides containing 30-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic
mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here,
we have synthesized 20-O-methyl-30-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme
reaction. We developed an efficient method for the synthesis of 20-O-methyl-30-thioguanosine phosphoramidite in eight steps starting
from 20-O-methyl-N2-(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-
phase synthesis, we used this new analogue to investigate whether the 30-oxygen of the guanosine cofactor in the Tetrahymena ribo-
zyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 20-hydroxyl group. We show that regardless of
whether the guanosine cofactor bears a 30-oxygen or 30-sulfur leaving group, replacing the adjacent 20-hydroxyl group with a 20-
methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen
bond donated by the guanosine 20-hydroxyl group contributes to catalytic function in a manner distinct from the U 20-hydroxyl
ꢀ1
group.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
for studying the chemistry of RNA splicing and the
principles of phosphoryl transfer reactions, crystallo-
graphic and biochemical analyses, including studies that
Oligonucleotides containing 30-S-phosphorothiolate
linkages (Fig. 1), in which a sulfur atom replaces the
30-bridging oxygen atom of the phosphodiester linkage,
provide valuable analogues for studying RNA structure
and function.1–14 Oligodeoxynucleotides containing this
linkage have been used to investigate the resolution of
the Holliday junction by RuvC, DNA repair by Esche-
richia coli DNA T:G mismatch endonuclease, and the
nucleolytic activity of E. coli DNA polymerase I and
the restriction endonuclease EcoRV.1,4,9–11,14,15 Phosp-
horothiolate linkages have also yielded fundamental in-
sights into the catalytic mechanisms of RNA enzymes
and RNA splicing machineries, revealing the participa-
tion of metal ions and hydrogen bonds in their catalytic
function.2,3,5,7,8,16–21 With respect to the self-splicing
group I intron, which has served as a model system
O
O
P
O
Base
O
O
S
H (or OH, OMe)
O
P
Base
O
O
O
O
H (or OH, OMe)
O
P
O
Keywords: Group I intron; RNA catalysis; Thionucleosides; Atomic
mutation cycle; 30-S-Phosphorothioamidite.
O
*
Corresponding author.
jpicciri@uchicago.edu
Fax:
+1
773
7020271; e-mail:
Figure 1. A 30-S-phosphorothiolate linkage.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.061

J. Lu et al. / Bioorg. Med. Chem. 16 (2008) 5754–5760
5755
group donates a hydrogen bond. We have used this
strategy with success to investigate the 20-hydroxyl
groups along the reaction coordinate in the Tetrahy-
mena ribozyme reaction. The cleavage site uridine
(Uꢀ1) and the guanosine nucleophile 20-hydroxyl groups
appear to donate hydrogen bonds in the transition state
of the reaction, yielding DGH removal values of 2.5 and
3.5 kcal/mol, respectively.27
H
N H
2 A207
O
G
U
22
H
A
O
MB
2
OH
O δ−
H
O
δ−
δ−
O
3
O
O
O
P
3
2
As the Uꢀ1 20-hydroxyl group donates a hydrogen bond
to the adjacent 30-oxygen atom in the transition state of
the ribozyme reaction (Fig. 2), we wanted to test
whether the analogous interaction involving the guano-
sine 20-hydroxyl group also occurs.28 AMC analysis also
offers a strategy to identify the corresponding hydrogen
bond acceptor. Mutations that disrupt a hydrogen bond
interaction between the 20-hydroxyl group and a poten-
tial acceptor should attenuate the cost of methoxy sub-
δ−
O
H
O
O
MA
G
MC
Figure 2. Transition state model of phosphoryl transfer catalyzed by
the Tetrahymena group I ribozyme. MA, MB, and MC represent the
catalytic metal ions in the active site. The red rectangle highlights the
20-hydroxyl group of the guanosine nucleophile under investigation.
Hatched lines indicate putative hydrogen bonds, and dots symbolize
metal ion coordination. Figure taken by permission from Ref. 27.
stitution (DDGOH ! OCH ) and, therefore, decrease the
3
value of DGH removal, as the ribozyme bearing the natural
nucleotide no longer gains a catalytic advantage from
hydrogen bond donation by the 20-hydroxyl group.27
To test whether the guanosine nucleophile 20-hydroxyl
group donates a hydrogen bond to the adjacent 30-oxy-
gen during the reaction, we plan to perform AMC anal-
ysis in the context of a 30-sulfur atom. Sulfur is a weak
hydrogen bond acceptor compared to oxygen.28–33
Therefore, if the guanosine 20-hydroxyl group donates
a hydrogen bond to the 30-oxygen during the reaction,
utilize phosphorothiolates, have revealed a complex net-
work of interactions within the active site of this ribo-
zyme (Fig. 2).22–24 Developing new methods to identify
and characterize these interaction networks remains
imperative to understanding catalysis by this and other
RNAs.
We previously devised an atomic mutation cycle (AMC)
to determine whether a RNA 20-hydroxyl group donates
a functionally important hydrogen bond (Fig. 3).25–27
AMC analysis requires the construction of three ana-
logues bearing modifications at the 20-position:
–OCH3, –NH2, and –NHCH3. When the energetic pen-
alty for the 20-OH to 20-OCH3 substitution
DDGOH ! OCH and DGH
should decrease in a
removal
3
30-sulfur background. To conduct AMC analysis, the
four 30-thioguanosine analogues corresponding to those
in Figure 3 must be prepared. As a starting point for this
analysis, here we describe the synthesis of one of these
analogues and its corresponding phosphoramidite, 20-
methoxy-30-thioguanosine.ꢀ Following solid-phase oli-
gonucleotide synthesis, we measured the reactivity of
this analogue in the Tetrahymena ribozyme reaction
and found that the energetic cost of methoxy substitu-
(DDGOH ! OCH ) exceeds that for the 20-NH2 to 20-
3
NHCH3 substitution (DDGNH ! NHCH3), we attribute
2
the difference to the absence of the hydrogen atom
(DGH removal) from the 20-OCH3 analogue. When the ab-
sence of the hydrogen atom engenders an energetic pen-
alty (DGH removal > 0), we infer that the 20-hydroxyl
tion (DDGOH ! OCH ) remains the same regardless of
3
the identity of 30-atom (oxygen/sulfur). These results
provide evidence against a hydrogen bond interaction
between the 20-hydroxyl group and the 30-oxygen atom
of the guanosine nucleophile.
2. Results and discussion
2.1. Synthesis of 20-O-methyl-30-thioguanosine
phosphoramidite
In 1999, Matulic-Adamic reported the synthesis of 30-
thioguanosine and its 30-phosphorothioamidite starting
from guanosine.34 The reaction of 50-N2-protected gua-
nosine with 2-acetoxyisobutyryl bromide afforded the
30-b-bromo-derivative stereoselectively, which was con-
verted to 30-S-acyl ribofuranosyl intermediates. The S-
acylated nucleosides were then converted in seven steps
ꢀ
The preparation of 30-thioguanosine was previously described.34,43
Figure 3. Atomic mutation cycle analysis of an RNA 20-hydroxyl
group.
The syntheses of 20-amino-30-thioguanosine and 20-methylamino-30-
thioguanosine are yet to be published.

5756
J. Lu et al. / Bioorg. Med. Chem. 16 (2008) 5754–5760
O
N
O
O
N
N
N
N
N
N
NH
NH
O
NH
O
O
i)
ii)
TBDPSO
N
TBDPSO
HO
N
N
H
N
N
O
O
O
H
H
90%
87%
HO OCH3
HO OCH3
TfO OCH3
1
2
3
O
N
O
N
O
N
N
N
N
N
N
NH
NH
NH
O
O
O
iv)
iii)
TBDPSO
TBDPSO
Br
TBDPSO
N
N
H
N
N
+
O
H
O
O
H
58%
H
O
AcS OCH3
OCH3
OCH3
O
H
4
6
O
5
O
N
N
N
N
N
NH
NH
NH
O
O
O
DMTrO
N
DMTrO
N
O
vii)
85%
v)
vi)
96%
N
N
N
N
HO
N
H
H
O
H
28%
two steps
AcS OCH3
HS OCH3
AcS OCH3
8
9
7
O
N
NH
O
viii)
DMTrO
N
N
N
O
H
quantitative
S
P
O
OCH3
CN
N
10
t
Scheme 1. Reagents and conditions: (i) BuPh2SiCl, Py, rt, 20 h; (ii) CF3SO2Cl, DMAP, CH2Cl2 0 ꢁC, 2 h; (iii) NaBr, CH3COCH3, reflux, 3 h; (iv)
KSAc, DMF, 60 ꢁC, 24 h; (v) Bu4NFÆ3H2O, AcOH, THF, rt, 36 h; (vi) (MeO)2TrCl, Py, rt, 24 h; (vii) Guanidine hydrochloride/NaOMe (4:1),
i
MeOH/CH2Cl2 (1:1), rt, 30 min; (viii) (iPr)2NP(Cl)OC2H4CN, Pr2NEt, 1-methyl-1H-imidazole, rt, 1 h.
to the 30-S-phosphorothioamidite in 13% overall yield
from guanosine. The corresponding 20-O-methyl-30-thi-
oguanosine phosphoramidite has not been reported.
We adapted Matulic-Adamic’s synthetic route to the
synthesis of 20-O-methyl-30-thioguanosine phospho-
ramidite 10.
tion of 7 as the 50-O-dimethoxytrityl ether was
performed in dry pyridine for 24 h by using excess
4,40-dimethoxytritylchloride (DMTrCl, 3 equiv), giving
50-DMTr protected compound 8 in 96% yield.
To prepare 30-phosphorothioamidite 10, the selective re-
moval of the 30-S-acetyl group of compound 8 must oc-
cur without the loss of the N2-isobutyryl group. A
solution of NaOMe and guanidine hydrochloride in
methanol proved to be effective.36 The treatment of 8
with a 4:1 mixture of guanidine hydrochloride and
25% NaOMe in methanol produced 30-SH derivative 9
in 85% yield. Phosphitylation of compound 9 under
standard conditions gave quantitative conversion to
30-phosphorothioamidite 10. We subsequently incorpo-
rated the 20-O-methyl-30-thioguanosine phosphorami-
Our synthesis begins with the commercially available
20-O-methyl-N2-isobutyrylguanosine 1 (Scheme 1). Reac-
tion of 1 with (tert-butyl)chlorodiphenylsilyl (tBuPh2-
SiCl) gave the corresponding 50-silyl derivative 2 in
90% yield. In the presence of 4-(dimethylamino)pyridine
(DMAP), the treatment of compound 2 with trifluoro-
methanesulfonyl chloride in CH2Cl2 at 0 ꢁC gave the
30-triflate derivative 3 in 83% yield.35 Subsequent SN2
substitution with NaBr in acetone afforded the desired
30-bromo derivative 4 in 58% yield. The treatment of 4
with KSAc in DMF at 60 ꢁC yielded a mixture of the de-
sired 30-S-acetyl derivative 6 and the 30,40-unsaturated
derivative 5, which forms via a competing elimination
0
0
dite into the oligonucleotide sequence, CUCG3 S,2 OCH A,
3
as previously described.43 Based on trityl yield data,
coupling efficiencies throughout oligonucleotide
synthesis were excellent (data not shown).
1
reaction.34 According to the H NMR spectra, 6 and 5
form in a 9:5 ratio. These two compounds could not
be separated by silica gel chromatography. We treated
the mixture of 5 and 6 with Bu4NFÆ3H2O and AcOH
in THF and subsequently isolated the desired 50-depro-
tected derivative 7 in 28% yield (two steps). During the
deprotection, the 30,40-unsaturated derivative decom-
posed and therefore could not be isolated. The protec-
2.2. Tetrahymena ribozyme reactions with substrates
bearing guanosine (G2 OH), 20-O-methylguanosine
0
(G2 OCH ), 30-thioguanosine (G3 S,2 OH), and 20-O-methyl-
0
0
0
3
30-thioguanosine (G3 S,2 OCH
)
0
0
3
Following oligonucleotide synthesis and deprotection, we
determined the effect of the 30-sulfur atom on 20-methoxy

J. Lu et al. / Bioorg. Med. Chem. 16 (2008) 5754–5760
5757
substitution in the Tetrahymena ribozyme reaction. We
utilized a reaction that mimics the second step of group
I intron self-splicing in which an oligonucleotide product
(CCCUCU) cleaves a specific phosphodiester bond of
CUCGA, a 30-splice site analogue (Eq. 1).37–41
Chemistry, University of California at Riverside, on the
VG-ZAB instrument.
4.1.1. 50-O-(tert-Butyl)diphenylsilyl-20-O-methyl-N2-iso-
butyrylguanosine (2). To a stirred solution of 20-O-
methyl-N2-isobutyrylguanosine 1 (2.02 g, 5.49 mmol) in
pyridine (20 mL), (tert-butyl)diphenylsilyl chloride
(2.25 mL, 8.23 mmol) was added under Ar. The mixture
was stirred at room temperature for 20 h, then quenched
with MeOH (4 mL), and evaporated to a syrup. The res-
idue was dissolved in CHCl3 (40 mL) and washed with
H2O. The organic layer was dried over anhydrous so-
dium sulfate. The sodium sulfate was filtered off, and
the solvent was removed by evaporation under vacuum.
The residue was purified by silica gel chromatography,
eluting with 2% methanol in chloroform, to give the
E
CCCUCU þ CUCGpA ! CCCUCUpA þ CUGG ð1Þ
0
We measured the rates of cleavage for CUCG2 OHA and
0
CUCG2 OCH A to obtain the cost of 20-methoxy substi-
3
tution in the context of a 30-oxygen atom. Under condi-
tions where chemistry is rate-limiting, 20-methoxy
substitution has a 6.9 kcal/mol (8.8 · 104-fold) effect on
reactivity (DDGOH ! OCH ) (Fig. 4). This effect remains
similar in the context of 3the 30-sulfur atom: under the
same conditions CUCG3 S,2 OCH A reacts 3.1 · 104-fold
0
0
3
0
0
slower than does CUCG3 S,2 OHA, indicating that the
replacement of the 20-OH group with a 20-OCH3 group
imposes a 6.2 kcal/mol effect on reactivity (DDGOH !
1
product as a white foam: 2.99 g (90% yield). H NMR
(CDCl3): d 12.16 (s, 1H), 9.10 (s, 1H), 7.97 (s, 1H),
7.65–7.33 (m, 10H), 5.94 (d, 1H, J = 5.0 Hz), 4.57 (t,
1H, J = 5.0 Hz), 4.17 (t, 1H, J = 5.0 Hz), 4.13 (m, 1H),
3.98 (dd, 1H, J = 2.5, 11.5 Hz), 3.85 (dd, 1H, J = 3.5,
11.5 Hz), 3.43 (s, 3H), 2.67 (m, 1H), 1.22 (d, 6H,
J = 6.5 Hz), 1.06 (s, 9H). 13C NMR (CDCl3): d 178.9,
155.5, 148.1, 147.8, 137.1, 135.6, 135.4, 132.6, 132.4,
130.0, 129.9, 127.9, 127.8, 121.4, 85.7, 85.1, 84.1, 69.3,
63.5, 58.8, 36.3, 26.9, 19.2, 19.0, 18.9. HRMS (FAB+):
m/z calcd for C31H40N5O6Si [M+H+]: 606.2748; found:
606.2760.
OCH ) (Fig. 4). If the 20-hydroxyl group were donating
3
a hydrogen bond to the adjacent 30-oxygen, sulfur sub-
stitution at the 30-position would be expected to disrupt
this interaction and therefore diminish the catalytic
advantage of the 20-OH group over the 20-OCH3 group.
However, as sulfur substitution only has a ꢁ3-fold
(ꢁ0.7 kcal/mol) effect on DDGOH ! OCH , our results
3
provide evidence against this interaction, suggesting that
another neighboring group must accept the hydrogen
bond from the 20-hydroxyl group.
4.1.2.
50-O-(tert-Butyl)diphenylsilyl-20-O-methyl-30-O-
trifluorosulfonyl-N2-isobutyryl guanosine (3). To a solu-
tion of 2 (606 mg, 1.0 mmol) and DMAP (244 mg,
2 mmol) in dry MeCN (6 mL), CF3SO2Cl (0.16 mL,
1.5 mmol) was added at 0 ꢁC under Ar. After the mix-
ture was stirred at 0 ꢁC for 2 h, the reaction was
quenched with ice water (5 mL) and the mixture was
stirred for 15 min. The reaction mixture was diluted with
CH2Cl2 (20 mL). The organic layer was separated, and
the aqueous layer was extracted with CH2Cl2 (2·
30 mL). The organic layers were combined and washed
with H2O and brine and subsequently dried over anhy-
drous sodium sulfate. The sodium sulfate was filtered
off, and the solvent was removed by evaporation under
vacuum. The residue was purified by silica gel chroma-
tography, eluting with 2% methanol in chloroform, to
give the product as a white foam: 644 mg (87% yield).
1H NMR (CDCl3): d 12.02 (s, 1H), 8.38 (s, 1H), 7.83
(s, 1H), 7.63–7.36 (m, 10H), 5.90 (d, 1H, J = 6.5 Hz),
5.75 (t, 1H, J = 4.0 Hz), 4.59 (t, 1H, J = 5.5 Hz), 4.38
(m, 1H), 3.96 (dd, 1H, J = 3.0, 12.0 Hz), 3.84 (dd, 1H,
J = 3.0, 12.0 Hz), 3.42 (s, 3H), 2.54 (m, 1H), 1.24 (d,
6H, J = 6.5 Hz), 1.04 (s, 9H). 13C NMR (CDCl3): d
178.3, 155.2, 148.1, 147.7, 137.0, 135.4, 135.3, 131.9,
131.8, 130.3, 130.2, 128.0, 121.7, 84.9, 83.2, 81.9, 81.3,
77.3, 77.0, 62.3, 59.4, 36.5, 26.7, 19.1, 18.9, 18.8. HRMS
(FAB+): m/z calcd for C32H39N5O8F3SiS, [M+H+]:
738.2210; found: 738.2235.
3. Conclusions
We established an efficient method for the synthesis of
20-O-methyl-30-thioguanosine phosphoramidite 10 in
10.4% overall yield starting from 20-O-methyl-N2-iso-
butyrylguanosine. Using solid-phase methods, we
achieved efficient incorporation of the 20-O-methyl-30-
thioguanosine phosphoramidite into RNA. We have
also utilized this analogue to investigate a potential
hydrogen bond interaction in the Tetrahymena ribozyme
reaction. Our results provide evidence against a model in
which the guanosine 30-oxygen accepts a hydrogen bond
from the adjacent 20-hydroxyl group. Thus, the hydro-
gen bond donated by the guanosine 20-hydroxyl group
contributes to catalytic function in a manner distinct
from the U 20-hydroxyl group. Further analysis will
ꢀ1
be required to identify its hydrogen-bonding partner.
4. Experimental
4.1. Chemistry
All reactions were carried out under a positive pressure
of Ar in anhydrous solvents. Commercially available re-
agents and anhydrous solvents were used without fur-
ther purification. H, 13C, and 31P NMR spectra were
1
50-O-(tert-Butyl)diphenylsilyl-20-O-methyl-30-b-
recorded on a Bruker 500 or Bruker 400 MHz NMR
spectrometer. 1H chemical shifts are reported in d
(ppm) relative to tetramethylsilane and 13C chemical
shifts d (ppm) relative to the solvent used. High resolu-
tion mass spectra were obtained from the Department of
4.1.3.
bromo-N2-isobutyryl-guanosine (4). A mixture of 3
(1.56 g, 2.12 mmol) and NaBr (0.87 g, 8.47 mmol) in
acetone (20 mL) was heated under reflux for 3 h under
Ar. The solvent was removed under reduced pressure,

5758
J. Lu et al. / Bioorg. Med. Chem. 16 (2008) 5754–5760
0
0
0
0
0
0
Figure 4. Ribozyme reactions with CUCG2 OHA, CUCG2 OCH A, CUCG3 S,2 OHA and CUCG3 S,2 OCH A. Reactions took place in the presence of
3
3
saturating ribozyme and product to form the EP complex. CUCGXA substrates were 50-32P-labeled (denoted by the asterisk). For ribozyme reactions
0
with the phosphorothiolate substrates, we observed that CUCG3 SH,2 OCH migrated slower than CUCG3 SH,2 OH. To confirm the presence of the free
0
0
0
3
0
thiol, we treated the ribozyme-catalyzed products with iodoacetamide, an alkylating sulfhydryl reagent (data not shown). Both CUCG3 SH,2 OH and
0
0
0
CUCG3 SH,2 OCH were susceptible to iodoacetamide modification and, upon alkylation, comigrated (data not shown).
3
and the residue was purified by silica gel chromatogra-
phy, eluting with 1–3% methanol in chloroform, to give
the product as a white foam: 0.822 g (58% yield). H
Following the removal of the solvent by evaporation un-
der vacuum, the resulting residue was purified by silica
gel chromatography, eluting with 2–4% methanol in
chloroform, to give product 7 as a yellow foam:
1
NMR (CDCl3): d 12.14 (s, 1H), 8.95 (s, 1H), 7.97 (s,
1H), 7.70–7.37 (m, 10H), 5.84 (d, 1H, J = 1.5 Hz), 4.41
(d, 1H, J = 3.5 Hz), 4.36 (m, 2H), 4.02 (dd, 1H,
J = 5.5, 10.5 Hz), 3.96 (dd, 1H, J = 6.0, 10.5 Hz), 3.47
(s, 3H), 2.66 (m, 1H), 1.23 (d, 6H, J = 5.5 Hz), 1.07 (s,
9H). 13C NMR (CDCl3): d 178.6, 155.6, 147.8, 147.7,
137.3, 135.5, 135.4, 132.8, 132.7, 130.0, 129.9, 127.8,
127.7, 121.2, 92.2, 88.8, 81.3, 64.5, 58.4, 50.2, 36.4,
26.8, 19.2, 19.0, 18.9. HRMS (FAB+): m/z calcd for
C31H39N5O5F3SiBr [M+H+]: 668.1898; found: 668.1913.
1
(137 mg, 28% from 4). H NMR: d 12.04 (s, 1H), 9.31
(s, 1H), 7.97 (s, 1H), 5.86 (d, 1H, J = 2.0 Hz), 5.06
(dd, 1H, J = 5.5, 9.0 Hz), 4.31 (d, 1H, J = 5.5 Hz), 4.21
(d, 1H, J = 9.0 Hz), 3.97 (d, 1H, J = 13.0 Hz), 3.72 (d,
1H, J = 13.0 Hz), 3.46 (s, 3H), 2.74 (m, 1H), 2.44 (s,
3H), 1.28 (d, 6H, J = 7.0 Hz). 13C NMR (CDCl3): d
196.2, 179.6, 155.4, 147.8, 147.3, 138.4, 121.2, 88.2,
85.1, 85.0, 60.8, 58.7, 43.4, 36.1, 30.6, 19.0, 18.8. HRMS
(FAB+): m/z calcd for C17H24N5O6S [M+H+]: 426.1447;
found: 426.1430.
4.1.4. 50-O-(tert-Butyl)diphenylsilyl-20-O-methyl-30-deoxy-
b-D-glycero-pent-30-enofuranosyl-N2-isobutyrylguanosine
(5) and 50-O-(tert-butyl) diphenyl-silyl-20-O-methyl-30-S-
acetyl-N2-isobutyrylguanosine (6). To a solution of 4
(49 mg, 0.073 mmol) in dry DMF (2 mL), potassium
thioacetate (25 mg, 0.22 mmol) was added, and the mix-
ture was stirred at 60 ꢁC for 24 h and evaporated to a
syrup. The residue was partitioned between an aqueous
NaHCO3 solution/brine (v/v, 1:1) and CH2Cl2; the or-
ganic layer was dried (Na2SO4) and evaporated. The res-
idue was purified by silica gel chromatography; eluting
with 1–2% methanol in chloroform, to give a mixture
of 5 and 6 (35 mg) (5/6 5:9 based on H NMR). H
NMR (CDCl3): d 12.08 (s, 1H, 5), 11.91 (s, 1H, 6),
9.24 (s, 1H, 6), 8.71 (s, 1H, 5), 7.99 (s, 1H, 5), 7.86 (s,
1H, 6), 7.28–7.70 (m, 20H, 5 + 6), 6.29 (d, 1H,
J = 1.6 Hz, 5), 5.89 (s, 1H, 6), 5.63 (m, 1H, 6), 5.43 (t,
1H, J = 1.1 Hz, 5), 4.66 (s, 1H, 5), 4.43 (d, 1H,
J = 4.7 Hz, 6), 4.29 (d, 1H, J = 9.0 Hz, 6), 4.13 (m, 1H,
5), 3.86 (dd, 1H, J = 2.1, 11.9 Hz, 6), 3.71 (dd, 1H,
J = 3.5, 11.9 Hz, 6), 3.50 (s, 3H, 6), 3.34 (s, 3H, 5),
2.67 (m, 1H, 5), 2.61 (m, 1H, 6), 2.44 (s, 3H, 6), 1.21–
1.27 (m, 12H, 5 + 6), 1.07 (s, 9H, 5), 0.88 (s, 9H, 6).
4.1.6.
50-O-(4,40-Dimethoxytrityl)-30-S-acetyl-20-O-
Methyl-N2-isobutyryl-30-thioguanosine (8). To a solution
of 7 (48 mg, 0.11 mmol) in dry pyridine (2 mL),
DMTrCl (115 mg, 0.34 mmol) was added. The mixture
was stirred at room temperature for 20 h, quenched with
MeOH, and evaporated to a syrup which was parti-
tioned between 5% aq NaHCO3 and CH2Cl2. The or-
ganic layer was washed with brine, dried (Na2SO4),
and evaporated, and the residue was purified by chro-
matography (silica gel, gradient 0–2%) MeOH/CH2Cl2)
with 0.5% Et3N to give compound 8 (79 mg, 96% yield).
1H NMR: d 7.88 (s, 1H), 7.15–7.31 (m, 9H), 6.71–6.73
(m, 4H), 5.90 (s, 1H), 5.58 (m, 1H), 4.36 (d, 1H,
J = 5.0 Hz), 4.17 (dd, 1H, J = 3.0, 11.0 Hz), 3.76 (s,
6H), 3.47 (s, 3H), 3.45 (m, 1H), 3.15 (m, 1H), 2.55 (m,
1H), 2.35 (s, 3H), 1.18 (d, 3H, J = 7.0 Hz), 1.12 (d,
3H, J = 7.0 Hz). 13C NMR (CDCl3): d 195.9, 178.7,
158.3, 155.6, 147.4, 147.3, 144.3, 138.7, 135.6, 135.5,
129.9, 128.0, 127.6, 126.7, 121.9, 112.82, 112.80. HRMS
(FAB+): m/z calcd for C38H41N5O8NaS [M+Na+]:
750.2568; found: 750.2594.
1
1
4.1.7. 50-O-(4,40-Dimethoxytrityl)-20-O-methyl-N2-isobu-
tyryl-30-thioguanosine (9). To a mixture of guanidine
hydrochloride (208 mg, 0.285 mmol), MeOH (5 mL)
and NaOCH3 (25% in MeOH, 60 lL) a solution of 8
(20 mg, 0.027 mmol) was added in CH2Cl2 (5 mL), and
the mixture was stirred at room temperature for 30 min.
The reaction mixture was partitioned between H2O and
CH2Cl2. The organic layer was dried over anhydrous so-
dium sulfate. The sodium sulfate was filtered off, and the
4.1.5. 30-S-Acetyl-20-O-methyl-N2-isobutyryl-30-thiogu-
anosine (7). To the above mixture 5/6 (0.90 g) in THF
(15 mL), AcOH (0.40 mL, 6.5 mmol) was added, fol-
lowed by Bu4NFÆ3H2O (0.80 g, 2.6 mmol). The mixture
was stirred at room temperature for 24 h, then diluted
with CH2Cl2, and washed with H2O and 10% aq NaH-
CO3. The aqueous layers were extracted with CH2Cl2
and the combined organic layers were dried (Na2SO4).

J. Lu et al. / Bioorg. Med. Chem. 16 (2008) 5754–5760
5759
solvent was removed by evaporation under vacuum. The
residue was purified by silica gel chromatography, eluting
with 2% methanol in chloroform with 0.5% Et3N, to give
compound 9 (16 mg, 85%). 1H NMR (CDCl3): d 12.26 (s,
1H), 9.59 (s, 1H), 8.04 (s, 1H), 7.17–7.42 (m, 9H), 6.72–
6.81 (m, 4H), 5.96 (d, 1H, J = 1.2 Hz), 4.04 (d, 1H,
J = 9.5 Hz), 4.00 (d, 1H, J = 4.5 Hz), 3.75 (s, 6H), 3.67
(m, 1H), 3.60 (d, 1H, J = 11.0 Hz), 3.48 (s, 3H), 3.38 (m,
1H), 2.58 (m, 1H), 1.71 (d, 1H, J = 10.0 Hz), 1.13 (d,
6H, J = 5.5 Hz). 13C NMR (CDCl3): d 179.3, 158.6,
155.7, 147.9, 147.6, 144.3, 137.0, 135.5, 135.4, 130.0,
129.9, 128.0, 127.9, 127.0, 121.6, 113.2, 87.2, 86.5, 86.2,
85.5, 61.0, 58.7, 55.2, 39.0, 36.1, 18.9, 18.8. HRMS
(FAB+): m/z calcd for C36H39N5O7NaS [M+Na+]:
708.2462; found: 708.2495.
stimulate cleavage of the phosphorothiolate substrates,
all reactions were performed in the presence of 10 mM
MnCl2. To ensure that chemistry was rate-limiting, reac-
0
0
0
tions with CUCG2 OHA and CUCG3 S,2 OHA were per-
formed with ꢀ1d,rP (CCCUCdT), a product analogue
with 20-deoxthymidine at the 30-terminus. This substitu-
tion slows the chemical step while having no effect on
other reaction steps.44 For the corresponding 20-methoxy
analogues, no reaction was observed with ꢀ1d,rP.
Therefore, reactions were performed in the presence of
an all-ribo product (CCCUCU) and the resulting kobs
was divided by 1188, the reported differential in reactivity
between rP and ꢀ1d,rP.44 Each rate constant represents
the average of two independent determinations that vary
by less than 25%.
4.1.8. 50-O-(4,40-Dimethoxytrityl)-20-O-methyl-N2-isobu-
tyryl-30-thioguanosine-30-S-(2-cyanoethyl)-N,N-diisopropyl-
phoramidothioite (10). To a solution of compound 9
(52 mg, 0.076 mmol) in dry dichloromethane (5 mL) un-
der Ar, were added N,N-diisopropylethylamine (66 lL,
0.34 mmol), 2-cyanoethyl N,N-diisopropylchlorophosph-
oramidite (51 lL, 0.34 mmol), and 1-methylimidazole
(6.2 lL, 0.076 mmol). The reaction mixture was stirred
at room temperature until all the starting material was
consumed (1 h). The reaction mixture was quenched with
methanol (1 mL) and stirred for 5 min. After the solvent
was removed, the residue was purified by silica gel chro-
matography, eluting with 2% acetone in dichloromethane
with 0.5% Et3N to give the corresponding phosphorami-
dite 10 (100% conversion). 31P NMR (CD3CN): d 167.0,
161.2. MS (FAB+): m/z calcd for C45H57N7O8PS
[M+H+]: 886.4; found: 886.2.
Acknowledgments
J.L. is a Research Associate, N.-S.L. is a Research Spe-
cialist, R.N.S. is a Research Technician, and J.A.P. is an
Investigator of the Howard Hughes Medical Institute.
We thank Q. Dai for assistance with MALDI mass spec-
trometry and C. Lea for assistance with substrate prep-
aration and kinetic measurements. We thank J. Ye, T.
Novak, J. Min De-Bartolo, S. Koo, and Q. Dai for help-
ful discussions and critical comments on the manuscript.
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