Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

Article abstract of DOI:10.1080/15257770.2014.965256

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The eff

Full text of DOI:10.1080/15257770.2014.965256

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Synthesis of 2′-O-Photocaged
Ribonucleoside Phosphoramidites
Jun Lua, Selene C. Kooa, Nan-Sheng Lia & Joseph A. Piccirillia
a Department of Biochemistry & Molecular Biology and Department
of Chemistry, University of Chicago, Chicago, Illinois, United States
Published online: 26 Jan 2015.
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To cite this article: Jun Lu, Selene C. Koo, Nan-Sheng Li & Joseph A. Piccirilli (2015) Synthesis of 2′-
O-Photocaged Ribonucleoside Phosphoramidites, Nucleosides, Nucleotides and Nucleic Acids, 34:2,
114-129, DOI: 10.1080/15257770.2014.965256
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Nucleosides, Nucleotides and Nucleic Acids, 34:114–129, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.965256
SYNTHESIS OF 2ꢀ-O-PHOTOCAGED RIBONUCLEOSIDE
PHOSPHORAMIDITES
Jun Lu, Selene C. Koo, Nan-Sheng Li, and Joseph A. Piccirilli
Department of Biochemistry & Molecular Biology and Department of Chemistry, University
of Chicago, Chicago, Illinois, United States
The chemical synthesis and incorporation of the phosphoramidite derivatives of 2ꢁ-O-photocaged
2
ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-
nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging
of these 2ꢁ-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl <
4,5-dimethoxy-2-nitrobenzyl < 2ꢁ-O-o-nitrobenzyl.
Keywords
2ꢁ-O-photocaged ribonucleosides; 2ꢁ-O-photocaged phosphoramdites;
2ꢁ-O-photocaged oligomers; 2ꢁ-O-o-nitrobenzyl; 2ꢁ-O-α-methyl-o-nitrobenzyl; 2ꢁ-O-4,5-
dimethoxy-2-nitrobenzyl.
INTRODUCTION
Photolabile protecting groups have been extensively investigated over
the years to protect hydroxyl or amino groups in organic synthesis.[1,2]
Photocaging provides a well-established strategy to cage functions of bi-
ologically important molecules until released by UV irradiation.[3–5] 2ꢁ-O-
Photocaged oligonucleotides including the 3ꢁ-S-oligonucleotide and 5ꢁ-S-
oligonucleotide phosphorothiolates as useful RNA analogues have been
successfully utilized to investigate the mechanism of RNA catalyzed re-
actions[6–10] and pre-mRNA splicing.[11,12] Although a number of photo-
labile groups have been introduced into photocaged molecules for bio-
logical studies, to the best of our knowledge, only o-nitrobenzyl group
has been introduced to 2ꢁ-O-caged RNAs.[6,10,13,14] For example, the 2ꢁ-O-
o-nitrobenzyl derivatives of adenosine,[15] cytidine,[15] guanosine,[16] and
uridine[17] were synthesized by Ohtsuka et al. and applied to the solution
synthesis of 2ꢁ-O-caged dinucleotides and trinucleotides.[15,17,18] The first
Received 30 July 2014; accepted 10 September 2014.
Address correspondence to Nan-Sheng Li or Joseph A. Piccirilli, Department of Biochemistry &
Molecular Biology and Department of Chemistry, University of Chicago, Chicago, Illinois, United States.
E-mail: nli@uchicago.edu or jpicciri@uchicago.edu
114

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
115
solid-phase synthesis of 2ꢁ-O-photocaged oligoribonucleotides was achieved
by using 2ꢁ-O-o-nitrobenzyl-3ꢁ-H-phosphonate chemistry.[13] Later, the phos-
phoramidite derivatives of 2ꢁ-O-o-nitrobenzyl-N 6-benzoyladenosine[6] and
2ꢁ-O-o-nitrobenzyl-N 4-benzoylcytidine[10] were prepared and applied to syn-
thesize the 2ꢁ-O-caged RNAs via phosphoramidite chemistry. These 2ꢁ-O-
o-nitrobenzyl RNAs have proven useful as tools to probe RNA reactivity
and dynamics via a photochemically-controlled manner.[6–12] The 2ꢁ-O-
photolabile group and ultramild N -phenoxyacetyl group protected phos-
phoramidites are useful for the synthesis of oligoribonucleotides contain-
ing a 5ꢁ-S-phosphorothiolate linkage since these modified oligoribonu-
cleotides are more susceptible to alkaline cleavage compared to the wild
type RNAs.[7,19] Only three 2ꢁ-O-photolabile phosphoramidite derivatives
(with 2ꢁ-O-o-nitrobenzyl and N -benzoyl or N -isobutyryl protection) have
been synthesized and reported in the literature.[10,14,20] 2ꢁ-O-o-Nitrobenzyl
caged RNAs synthesized by either solid phase synthesis or a two-step liga-
tion method have been successfully applied to investigate the mechanisms
of ribozyme catalyzed reactions.[7–9,19] However, the relatively long UV ir-
radiation time (4 to 6 minutes)[9,19] for the full removal of o-nitrobenzyl
group from 2ꢁ-O-photocaged RNAs may limit its applications in the case of
unstable RNAs (or ribozymes) with UV exposure for a long time. To over-
come this limitation, here we report the general synthetic methods to obtain
2ꢁ-O-photocaged phosphoramidites containing a number of more efficient
2ꢁ-O-photolabile and N -phenoxyacetyl protected ribonucleoside phosphora-
midites.
RESULTS AND DISCUSSION
The 2ꢁ-O-photolabile 5ꢁ-O-DMTr-N 2-butyrylguanosine derivatives (2a and
2b) were prepared in 21% and 18% yields, respectively, according to our
previously improved procedures by the reactions of N 2-butyrylguanosine
(1a) with o-nitrobenzyl bromide or α-methyl-o-nitrobenzyl bromide, followed
by the 5ꢁ-O-DMTr protection.[16,19] The corresponding 2ꢁ-O-photolabile 5ꢁ-
O-DMTr-N 2-phenoxyacetylguanosine derivatives (2c and 2d) were prepared
from N 2-phenoxyacetylguanosine (1b) in 20% and 5% yields, respectively
(Scheme 1). It is worthy to note the free guanosine could not be selectively
2ꢁ-O-benzylated by the reaction with o-nitrobenzyl bromide due to the side
reaction at N1 of the guanine ring.
The 2ꢁ-O-photolabile 5ꢁ-O-DMTr-N -phenoxyacetyl adenosine and cyti-
dine derivatives (6a–6d) could be prepared from their respective free ribonu-
cleoside precursors (i.e. adenosine and cytidine) in three steps (Scheme 2).
Without any protection both adenosine and cytidine could be selectively
2ꢁ-O-benzylated with 4,5-dimethoxy-2-nitrobenzyl bromide or α-methyl-o-
nitrobenzyl bromide to give 4a-4d. The amino group of nucleobases of

116
Jun Lu et al.
O
N
O
N
N
N
1. NaH/DMF
O2N
Br
O
NH
N
N
O
NH
R
N
DMTrO
R
H
N
HO
H
O
O2N
O
X
OH
O
2. DMTrCl/Py
OH OH
X
1a: R = CH(CH3)2
1b: R = CH2OPh
2a: R = CH(CH3)2, X = H, 21% yield
2b: R = CH(CH3)2, X = Me, 18% yield
2c: R = CH2OPh, X = H, 20% yield
2d: R = CH2OPh, X = Me, 5% yield
SCHEME 1
Y
Br
HO
B
Z
O
1. TMSCl/Py
Y
HO
B
X
2. PhOCH2COCl
O
O2N
3. NH4OH
OH
O
Z
OH OH
NaH/DMF
X
O2N
3a: B = A
3b: B = C
4a: X = H, Y = Z = OMe,
B = A, 54% yield
4b: X = Me,Y = Z = H,
B = A
4c: X = Y = Z = H,
B = C, 18% yield (Ref. 15)
4d: X = Me,Y = Z = H,
B = C
DMTrO
B
HO
B
X
O
O
Y
Y
Z
DMTrCl/Py
OH
O
OH
O
Z
X
O2N
O2N
6a: X = H, Y = Z = OMe,
B = APhOAc, 72% yield
6b: X = Me,Y = Z = H,
B = APhOAc, 67% yield
6c: X = Y = Z = H,
5a: X = H, Y = Z = OMe,
B = APhOAc, 91% yield
5b: X = Me,Y = Z = H,
B = APhOAc, 65% yield from 3a
5c: X = Y = Z = H,
B = CPhOAc, 86% yield
6d: X = Me, Y = Z = H,
B = CPhOAc, 71% yield
5d: X = Me,Y = Z = H,
B = CPhOAc
B = CPhOAc, 14% yield from 3b
SCHEME 2

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
117
O
N
O
O
N
1. n-Bu2SnO/MeOH
NH
NH
NH
O2N
O
N
O
O
Br
2.
HO
HO
DMTrO
DMTrCl/Py
75%
O
O
O
23% in two steps
(Ref. 17 and 21)
OH OH
OH
5e
O
OH O
NO2
NO2
3c
6e
SCHEME 3
4a–4d was then selectively protected by a phenoxyacetyl group to give
compounds 5a–5d. 5ꢁ-O-DMTr protection of 5a–5d gave the correspond-
ing phophoramidite precursors 6a–6d in three steps from free nucleosides
3a and 3b with 11–44% overall yields.
Ohtsuka and Ikehara reported the synthesis of 2ꢁ-O-o-nitrobenzyluridine
(5e) in 24% yield by the reaction of 2ꢁ,3ꢁ-O-(dibutylstannyl)uridine[21]
with o-nitrobenzyl bromide in DMF at 110◦C for 4 hours (Scheme 3).[17]
DMTr protection of 5e then yielded the corresponding 2ꢁ-O-photocaged
uridine phosphoramidite precursor 6e in 75% yield. Unfortunately, we
found this method cannot be extended for the preparation of 2ꢁ-O-α-methyl-
o-nitrobenzyluridine by reacting of 2ꢁ,3ꢁ-O-(dibutylstannyl)uridine with α-
methyl-o-nitrobenzyl bromide. No desired product was formed after this
reaction, even at 110◦C for 24 hours. According to the procedure for the
synthesis of 2a, direct deprotonation of uridine with NaH in DMF, followed
by reaction with o-nitrobenzyl bromide generated only O4-o-nitrobenzyl-
uridine.
Phosphitylation of 2a–3d and 6a–6e with 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite yielded the corresponding 2ꢁ-O-
photocaged nucleoside phosphoramidites 7a–7i including the 2ꢁ-O-
photolabile N -phenoxyacetylribonucleoside phosphoramidites 7c–7h in
good yields (Scheme 4 & Table 1).
DMTrO
DMTrO
O
B
B
O
O
i-Pr2NP(Cl)OCH2CH2CN/
1-methyl-1H-imidazole/
i-Pr2NEt
Y
Y
OH
O
O
O
Z
P
Z
63-95%
X
X
N(Pr-i)2
NC
O2N
2a-2d, 6a-6f
SCHEME 4
O2N
7a-7j
To compare the efficiency of the UV irradiated decaging of RNAs
containing various photolabile groups, the known phosphoramidite
derivative of 2ꢁ-O-o-nitrobenzyl-N 6-benzoyladenosine 7j was also prepared

118
Jun Lu et al.
TABLE 1 Phosphoramidites 7a-7j prepared by the phosphitylation of 2a-2d and 6a-6f
Product
Precursor
X
Y
Z
B
Yield (%)
7a
7b
7c
7d
7e
7f
7g
7h
7i
2a
2b
2c
2d
6a
6b
6c
6d
6e
6f
H
Me
H
Me
H
Me
H
Me
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
Gi-Bu
Gi-Bu
95
86
77
63
88
92
92
91
86
82
GPhOAc
GPhOAc
APhOAc
APhOAc
CPhOAc
CPhOAc
U
7j
H
ABz
(Scheme 4).[6,14] The adenosine phosphoramidites 7e (with 3,4-dimethoxy-
2-nitrobenzyl group), 7f (with α-methyl-o-nitrobenzyl group), and 7j (with o-
nitrobenzyl group) were then incorporated into a 30-mer RNA sequence- (an
oligomer fragment for the construction of glms ribozyme):[22] 5ꢁ-GGU AAA
UUA UAG AXG CGC CAG AAC UAC ACC-3ꢁ, where X represented the cou-
pling of 2ꢁ-O-photocaged phosphoramidites (7e, 7f or 7h) at the ribozyme
cleavage site. The solid-phase synthesis was carried out on Expedite 8900 Nu-
cleic Acid Synthesizer with a modified RNA 1 μmole protocol. The standard
RNA 1 μmole protocol for X was modified to double coupling before capping
and oxidation. The trityl yields for the coupling of 7e, 7f or 7h were com-
parable to the commercially available phosphoramidites. After deprotection
with ammonium hydroxide, and desilylation with 1-methyl-2-pyrrolidinone
(NMP)/Et3N/Et3N-3HF,[23] these RNAs were 5ꢁ-radiolabeled and purified by
dPAGE gel as usual.[19] The progress of UV irradiated decaging of these RNAs
was analyzed after alkaline hydrolysis and quantified by phosphorimager ac-
cording to our reported method.[9,19] As expected, no alkaline hydrolysis was
observed at the caged nucleotide site until UV irradiation (Figure 1). These
data were then fitted into a first-order kinetics equation (y = y0 – A∗e-kt); and
the resulting kinetics rates are shown in Table 2. From Table 2, we conclude
that 2ꢁ-O-caged RNA containing an α-methyl-o-nitrobenzyl group has the
most prominent photolabile properties. An RNA oligomer with α-methyl-o-
nitrobenzyl group is uncaged by UV at 365 nm about 10 times faster com-
pared to an RNA oligomer with o-nitrobenzyl group, whereas an RNA strand
with 3,4-dimethoxy-2-nitrobenzyl group is uncaged by UV about 5 times faster
than an RNA oligomer with o-nitrobenzyl group at 365 nm. The electronic
donating groups (α-methyl and 3,4-dimethoxy) may have stabilized the res-
onance isomer (intermediate) formation thus facilitating the UV irradiated
deprotection.[24]

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
119
FIGURE 1 UV initiated deprotection and alkaline hydrolysis of 2ꢁ-O-caged RNAs containing 2ꢁ-O-
photolabile groups. A: Gel image corresponds to the RNA containing α-methyl-o-nitrobenzyl protecting
group. B: Gel image corresponds to the RNA containing o-nitrobenzyl protecting group. Method: A
5ꢁ-radiolabeled 30 mer RNA fragment in water (20 μL) was irradiated at 365 nm and 0.5-μL aliquots
were taken and diluted in water (3 μL). Alkaline hydrolysis ladders were performed by adding sodium
bicarbonate, pH 9, and incubating at 90 ◦C for 15 minutes. The resulting ladder of RNA fragments was
separated by denaturing PAGE, and deprotection was quantitated by measuring the intensity of the band
corresponding to cleavage by the deprotected nucleophile. The first lane in the depicted gel corresponds
to deprotected substrate prior to alkaline hydrolysis.
In summary, we have described the synthesis of the phospho-
ramidite derivatives of adenosine, cytidine and guanosine with more
2ꢁ-O-photolabile and N -phenoxyacetyl protections. We also prepared the
phosphoramidite derivative of 2ꢁ-O-o-nitrobenzyluridine. Solid phase syn-
thesis using these phosphoramidites together with commercially available
2ꢁ-O-TBS-N -phenoxyacetylribonucleoside phosphoramidites would provide
a general method to synthesize 2ꢁ-O-caged RNA oligonucleotides featured
with ultramild deprotection conditions (NH4OH/rt, 2 hours). Application
of this new class of ribonucleoside phosphoramidites in the synthesis of

120
Jun Lu et al.
TABLE 2 Efficiency of the UV (365 nm) irradiated uncaging of 2ꢁ-O-caged RNAs containing various
2ꢁ-O-photolabile groups
a
2ꢁ−O-Caged RNAs
k (min−1
)
t1/2 (min)b
Time (min)c
2ꢁ-O-α-methyl-o-nitrobenzyl-RNA
2ꢁ-O-2-nitro-3,4-(MeO)2C6H3CH2-RNA
2ꢁ-o-nitrobenzyl-RNA
3.0
1.6
0.27
0.23
0.43
2.6
0.83
1.5–3
4–6
a k is obtained from alkaline hydrolysis data of UV initiated uncaging of RNAs by fitting to the equation:
y = y0 – A∗e-kt
.
bt1/2 is the time when 50% RNAs are uncaged.
cTime estimated for full uncaging.
RNA oligomers containing a 2ꢁ-O-photocaged-5ꢁ-S-phosphorothiolate link-
age are currently being further evaluated and the syntheses are expected to
be reported elsewhere later. Overall, 2ꢁ-O-photocaged RNAs containing an
α-methyl-o-nitrobenzyl group seems to be the most photolabile amongst the
three photocaged groups studied.
EXPERIMENTAL SECTION
5ꢁ-O-(Dimethoxytrityl)-N2-isobutyryl-2ꢁ-O-(o-nitrobenzyl)guanosine
(2a).[19] Compound 2a was prepared from N 2-isobutyrylguanosine (1a) in
two steps (21% yield). Under argon N 2-Isobutyrylguanosine (1a) (1.717 g,
4.86 mmol) was treated with NaH (292 mg, 12.2 mmol) in dry DMF
(40 mL) at 0◦C. After hydrogen gas generation ceased (about 40 minutes),
o-nitrobenzyl bromide (1.575 g, 7.29 mmol) was added and the mixture was
stirred for 5 h at 0◦C. The reaction was quenched with EtOH and neutralized
with 1 N HCl. The mixture was evaporated and the crude product was
purified by silica gel chromatography, eluting with 4–6% methanol in
dichloromethane, to give N 2-isobutyryl-2ꢁ-O-(o-nitrobenzyl)guanosine[16,19]
as a yellow foam: 0.76 g (32% yield). To a solution of N 2-isobutyryl-2ꢁ-O-
(o-nitrobenzyl)guanosine (192 mg, 0.393 mmol) in dry pyridine (4.0 mL)
under dry argon, DMTrCl (400 mg, 1.18 mmol) was added. After the
mixture was stirred at rt overnight, methanol (2.0 mL) was added to quench
the reaction. The solvent was removed by rotary evaporation, and the crude
product was purified by silica gel chromatography, eluting with 2% MeOH
in CH2Cl2 containing 0.5% Et3N to give compound 2a[19] as a yellow foam
1
(206 mg, 66% yield). H NMR (CDCl3) δ 12.02 (br s, 1H), 8.61 (br s, 1H),
8.01 (d, J = 8 Hz, 1H), 7.87 (s, 1H), 7.67–7.18 (m, 12H), 6.8 (m, 4H), 6.01
(d, J = 3.5 Hz, 1H), 5.37 (d, J = 15 Hz, 1H), 5.22 (d, J = 15 Hz, 1H), 4.62
(t, J = 4.5 Hz, 1H), 4.55 (m, 1H), 4.27 (m, 1H), 3.77 (s, 3H), 3.767 (s, 3H),
3.55 (d, J = 10.5 Hz, 3H), 3.35 (m, 1H), 3.21 (br s, 1H), 2.29 (m, 1H), 1.12
(d, J = 7 Hz, 1H), 1.01 (d, J = 7 Hz, 1H); 13C NMR (DMSO-d6) δ 231.0,
178.8, 158.6, 155.5, 147.5, 147.4, 146.9, 144.6, 137.5, 135.8, 135.6, 134.1,

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
121
133.8, 130.0, 129.9, 128.6, 128.5, 128.0, 127.9, 127.0, 124.7, 121.9, 113.2,
113.1, 87.0, 86.4, 83.6, 82.3, 69.4, 69.3, 63.1, 55.2, 36.2, 18.7, 18.6; HRMS
calcd for C42H43N6O10 [MH+] 791.3035, found 791.3041.
5ꢁ-O-(Dimethoxytrityl)-N2-isobutyryl-2ꢁ-O-(α-methyl-o-nitrobenzyl)guano-
sine (2b).[19] According to the procedure described for the synthesis
of 2a, N 2-isobutyryl-2ꢁ-O-(α-methyl-o-nitrobenzyl)guanosine[19] (636 mg,
26% yield) as a yellow foam was prepared from 1a (1.717 g, 4.86 mmol),
NaH (292 mg, 12.2 mmol) and α-methyl-o-nitrobenzyl bromide (1.677 g,
7.29 mmol). Compound 2b was then prepared by the reaction of N 2-
isobutyryl-2ꢁ-O-(α-methyl-o-nitrobenzyl)guanosine (307 mg, 0.61 mmol)
with DMTrCl (621 mg, 1.83 mmol) in dry pyridine at rt overnight. Silica
gel chromatography (1–2% methanol in dichloromethane containing 0.5%
triethylamine) of the crude product yielded 2b[19] (345 mg, 70% yield) as
1
a yellow foam. H NMR (CDCl3) δ 12.04 (s, 1H), 8.57 (s, 1H), 7.92–7.70
(m, 3H), 7.47–7.19 (m, 10H), 6.82–6.79 (m, 4H), 6.01 (d, J = 1.2 Hz, 1H),
5.77 (m, 1H), 4.33–4.26 (m, 2H), 3.91 (m, 1H), 3.770 (s, 3H), 3.767 (s,
3H), 3.57–3.45 (m, 2H), 2.72 (d, J = 8.3 Hz, 1H), 2.60 (m, 1H), 1.66 (d,
J = 6.3 Hz, 3H), 1.27 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 6.8 Hz, 3H); 13C
NMR (CDCl3) δ 178.8, 158.5, 155.3, 147.8, 147.6, 147.0, 144.4, 139.0, 136.6,
135.6, 135.5, 134.7, 130.0, 128.8, 128.0, 127.9, 127.0, 124.6, 121.9, 113.2,
87.6, 86.6, 82.7, 80.4, 72.4, 69.1, 62.5, 55.2, 36.5, 24.1, 19,2, 18.4; HRMS
calcd for C43H45N6O10 [MH+] 805.3192, found 805.3209.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-N2-phenoxyacetylguanosine
(2c). According to the procedure described for the synthesis of 2a,
2ꢁ-O-(o-nitrobenzyl)-N 2-phenoxyacetylguanosine (0.81 g, 30% yield) as
a yellow foam was prepared from N 2-phenoxyacetylguanosine (1b)[25]
(2.027 g, 4.86 mmol), NaH (292 mg, 12.2 mmol) and o-nitrobenzyl bromide
(1.575 g, 7.29 mmol). Compound 2c was then prepared by the reaction
of 2ꢁ-O-(o-nitrobenzyl)-N 2-phenoxyacetylguanosine (308 mg, 0.56 mmol)
with DMTrCl (565 mg, 1.68 mmol) in dry pyridine at rt overnight. Silica
gel chromatography (1–2% methanol in dichloromethane containing 0.5%
triethylamine) of the crude product yielded 2c (317 mg, 67% yield) as a
yellow foam. 1H NMR (DMSO-d6) δ 8.11 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H),
7.73–7.69 (m, 2H), 7.55 (t, J = 6.6 Hz, 1H), 7.35–7.20 (m, 11H), 7.00–6.96
(m, 3H), 6.85–6.81 (m, 4H), 6.06 (d, J = 5.2 Hz, 1H), 5.47 (d, J = 2.6 Hz,
1H, disappeared with D2O), 5.06 (d, J = 14.7 Hz, 1H), 4.97 (d, J = 14.6 Hz,
1H), 4.85 (d, J = 2.5 Hz, 2H), 4.59 (t, J = 5.1 Hz, 1H), 4.42 (m, 1H), 4.12
(m, 1H), 3.72 (s, 6H), 3.28–3.18 (m, 2H); 13C NMR (CDCl3) δ 169.7, 158.6,
156.6, 147.7, 147.4, 144.3, 136.8, 135.5, 135.4, 133.8, 133.1, 130.0, 129.8,
128.9, 128.7, 128.1, 127.9, 127.0, 124.7, 122.6, 122.0, 114.8, 113.2, 86.7, 86.3,
83.6, 82.7, 69.8, 69.3, 67.0, 63.0, 55.2; HRMS calcd for C46H43N6O11 [MH+]
855.2984, found 855.2996.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N2-phenoxyacetyl-
guanosine (2d). According to the procedure described for the synthesis

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Jun Lu et al.
of 2a, a crude 2ꢁ-O-(α-methyl-o-nitrobenzyl)-N 2-phenoxyacetylguanosine
(530 mg, 19% yield) with some impurity as a yellow foam was prepared
from 1b (2.09 g, 5.01 mmol), NaH (301 mg, 12.54 mmol) and α-methyl-o-
nitrobenzyl bromide (1.729 g, 7.52 mmol). Compound 2d was then prepared
by the reaction of 2ꢁ-O-(α-methyl-o-nitrobenzyl)-N 2-phenoxyacetylguanosine
(107 mg, 0.19 mmol) with DMTrCl (192 mg, 0.57 mmol) in dry pyridine at
rt overnight. Silica gel chromatography (1% methanol in dichloromethane
containing 0.5% triethylamine) of the crude product yielded 2d (47 mg,
28% yield) as a yellow foam. 1H NMR (DMSO-d6) δ 7.76 (d, J = 7.8 Hz, 1H),
7.70 (s, 1H), 7.45–7.17 (m, 15H), 7.07–6.74 (m, 7H), 5.80 (d, J = 4.7 Hz,
1H), 5.43 (m, 1H), 4.63 (s, 2H), 4.53 (t, J = 4.8 Hz, 1H), 4.40 (t, J = 4.8 Hz,
1H), 4.21 (m, 1H), 3.75 (s, 6H), 3.42 (dd, J = 2.4, 10.7 Hz, 1H), 3.35 (dd,
J = 2.4, 10.7 Hz, 1H), 1.59 (d, J = 6.3 Hz, 3H); 13C NMR (CDCl3) δ 169.8,
158.5, 156.7, 147.4, 144.3, 138.7, 137.4, 135.5, 133.8, 129.9, 129.9, 129.7,
128.6, 128.0, 127.8, 127.4, 126.9, 123.8, 122.5, 114.7, 113.1, 86.5, 85.9, 84.1,
81.1, 73.9, 69.4, 67.0, 63.3, 55.1, 23.7; HRMS calcd for C47H45N6O11 [MH+]
869.3141, found 869.3158.
2ꢁ-O-(4,5-Dimethoxy-2-nitrobenzyl)adenosine (4a). According to the pro-
cedure described for the synthesis of 2a, compound 4a was prepared
from adenosine (1.51 g, 5.43 mmol), NaH (196 mg, 8.17 mmol) and 4,5-
dimethoxy-2-nitrobenzyl bromide (2.248 g, 8.14 mmol). Silica gel chro-
matography (0–8% methanol in dichloromethane) of the crude product
yielded 4a (1.41 g, 54% yield) as a yellow foam. 1H NMR (DMSO-d6) δ 8.35
(s, 1H), 8.06 (s, 1H), 7.56 (s, 1H), 7.35 (brs, 2H), 7.20 (s, 1H), 6.13 (d, J =
5.0 Hz, 1H), 5.50 (d, J = 5.0 Hz, 1H), 5.42 (m, 1H), 5.07 (d, J = 15.0 Hz,
1H), 4.87 (d, J = 15.0 Hz, 1H), 4.56 (t, J = 5.0 Hz, 1H), 4.40 (m, 1H), 4.05
(m, 1H), 3.82 (s, 3H), 3.71 (s, 3H), 3.70 (m, 1H), 3.59 (m, 1H); 13C NMR
(DMSO-d6) δ 156.1, 153.2, 152.4, 148.9, 147.3, 139.5, 139.0, 129.4, 119.3,
110.1, 107.8, 86.3, 86.1, 81.3, 68.9, 67.9, 61.3, 56.0, 55.8; HRMS calcd for
C19H23N6O8 [MH+] 463.1572, found 463.1560.
2ꢁ-O-(4,5-Dimethoxy-2-nitrobenzyl)-N6-phenoxyacetyladenosine
(5a).
Compound 4a (0.68 g, 1.47 mmol) was dried by co-evaporation with dry
pyridine (3 × 50 mL) under vacuum, and resuspended in dry pyridine
(30 mL) under Ar. Chlorotrimethylsilane (1.5 mL) was added to the
suspension at 0◦C. After the reaction mixture was stirred at rt for 45
minutes, a solution of phenoxyacetyl chloride (310 μL, 2.21 mmol) and
1,2,4-triazole (155 mg, 2.26 mmol) in dry pyridine/acetonitrile (20 mL,
1:1) was slowly added. After the mixture was stirred at 55◦C overnight, it was
cooled to rt, and the reaction was quenched by addition of H2O (1.5 mL).
After stirring for 5 minutes, concentrated aqueous NH4OH (1 mL) was
added at 0◦C, and the mixture was stirred for 30 minutes. The solvent was
removed, the crude product was purified by silica gel chromatography,
eluting with 3–5% MeOH in CH2Cl2 to give 5a (797 mg, 91% yield) as
1
a yellow foam. H NMR (CDCl3) δ 9.67 (br s, 1H), 8.71 (s, 1H), 8.17 (s,

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
123
1H), 7.44 (s, 1H), 7.37–7.33 (m, 2H), 7.07–7.04 (m, 3H), 6.76 (s, 1H),
6.07 (d, J = 7.3 Hz, 1H), 5.03 (d, J = 12.8 Hz, 1H), 4.96–4.93 (m, 3H),
4.71–4.65 (m, 2H), 4.40 (s, 1H), 3.99 (dd, J = 1.5, 13.0 Hz, 1H), 3.87 (s,
3H), 3.82 (s, 3H), 3.81 (dd, J = 1.5, 13.0 Hz, 1H); 13C NMR (CDCl3) δ
167.3, 157.1, 153.1, 152.0, 150.4, 149.1, 148.5, 143.7, 140.2, 129.9, 129.7,
126.7, 123.8, 115.0, 114.6, 111.4, 108.1, 89.1, 88.1, 81.2, 70.7, 70.1, 68.3,
65.1, 63.1, 56.5, 56.4; HRMS calcd for C27H29N6O10 [MH+] 597.1940, found
597.1937.
2ꢁ-O-(o-Nitrobenzyl)-N4-phenoxyacetylcytidine (5c).[19] 2ꢁ-O-(o-Nitro-
benzyl)cytidine (4c)[15] (3.58 g, 9.46 mmol) was dried by co-evaporation
with dry pyridine (3 × 100 mL) under vacuum and resuspended in dry
pyridine (180 mL) under argon. Chlorotrimethylsilane (9.0 mL) was added
to the suspension at 0◦C. After the mixture was stirred at rt for 45 minutes, a
solution of phenoxyacetyl chloride (2.00 mL, 14.4 mmol) and 1,2,4-triazole
(0.980 g, 14.3 mmol) in pyridine-acetonitrile (120 mL, 1:1) was slowly
added. After the mixture was stirred at 55◦C overnight, it was cooled to rt,
and the reaction was quenched by addition of H2O (10 mL). After stirring
for 5 minutes, concentrated aqueous NH4OH (6.5 mL) was added at 0◦C,
and the mixture was stirred for 30 minutes. The solvent was removed, and
the crude product was purified by silica gel chromatography, eluting with
3–5% MeOH in CH2Cl2 to give 5c[19] (3.44 g, 71% yield) as a yellow foam.
1H NMR (DMSO-d6) δ 11.04 (s, 1H), 8.51 (d, 1H, J = 7.5 Hz), 8.08 (dd,
1H, J = 1.3, 8.2 Hz), 7.94 (d, 1H, J = 7.7 Hz), 7.77 (m, 1H), 7.57 (m, 1H),
7.35–7.25 (m, 2H), 7.11 (d, 1H, J = 7.5 Hz), 6.96 (m, 1H), 6.92 (m, 2H),
5.94 (d, 1H, J = 2.4 Hz), 5.34 (d, 1H, J = 6.7 Hz), 5.25 (t, 1H, J = 5.0 Hz),
5.15 (s, 2H), 4.83 (s, 2H), 4.12 (m, 1H), 4.15 (s, 1H), 4.03–3.95 (m, 3H),
3.79 (m, 1H), 3.64 (m, 1H); HRMS calcd for C24H25N4O9 [MH+] 513.1622,
found 513.1621.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(4,5-dimethoxy-2-nitrobenzyl)-N6-phenoxy-
acetyladenosine (6a). According to the procedure described for the
synthesis of 2a, compound 6a was prepared by the reaction of 5a (797 mg,
1.34 mmol) with DMTrCl (1.25 g, 4.02 mmol) in dry pyridine at rt overnight.
Silica gel chromatography (1% methanol in dichloromethane containing
0.5% triethylamine) of the crude product yielded 6a (864 mg, 72% yield)
1
as a yellow foam. H NMR (CDCl3) δ 9.47 (brs, 1H), 8.66 (s, 1H), 8.26 (s,
1H), 7.59 (s, 1H), 7.44–6.81 (m, 19H), 6.30 (d, J = 4.1 Hz, 1H), 5.24 (d, J =
14.0 Hz, 1H), 5.08 (d, J = 14.0 Hz, 1H), 4.86 (s, 2H), 4.74 (t, J = 4.6 Hz,
1H), 4.57 (t, J = 4.9 Hz, 1H), 4.32 (m, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.78
(s, 6H), 3.57 (dd, J = 3.2, 10.8 Hz, 1H), 3.46 (dd, J = 4.1, 10.7 Hz, 1H), 2.92
(b, 1H); 13C NMR (CDCl3) δ 158.7, 157.1, 153.6, 152.6, 151.4, 148.4, 148.3,
144.5, 141.9, 140.1, 135.62, 135.60, 130.2, 130.0, 128.2, 128.0, 127.8, 127.1,
123.2, 122.5, 115.0, 113.3, 110.6, 108.2, 87.1, 86.9, 84.3, 81.9, 70.1, 69.8,

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Jun Lu et al.
68.2, 62.9, 56.5, 56.4, 55.3; HRMS calcd for C48H47N6O12 [MH+] 899.3246,
found 899.3244.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N6-phenoxyacetyl-
adenosine (6b). According to the procedure described for the synthesis of
2a, a crude 2ꢁ-O-(α-methyl-o-nitrobenzyl)adenosine (4b) (1.42 g) as a yellow
foam was prepared from adenosine (2.00 g, 7.48 mmol), NaH (269 mg,
11.2 mmol) and α-methyl-o-nitrobenzyl bromide (2.51 g, 10.9 mmol).
Compound 5b was then prepared from the crude 4b (1.42 g, 3.41 mmol),
TMSCl (3.5 mL), and phenoxyacetyl chloride (0.71 mL, 5.12 mmol) as
described for the synthesis of 5a. Silica gel chromatography (5% methanol
in dichloromethane) of the crude product yielded 5b (1.20 g, 65% yield,
1
two steps from adenosine) as a yellow foam. H NMR (CDCl3) δ 9.55 (s,
1H), 8.71 (s, 1H), 8.13 (s, 1H), 7.72–7.06 (m, 9H), 6.01 (d, J = 7.0 Hz, 1H),
5.42 (m, 1H), 5.11 (m, 1H), 4.93 (s, 2H), 4.85 (m, 1H), 4.40 (d, J = 4.6 Hz,
1H), 4.32 (d, J = 1.3 Hz, 1H), 3.90 (m, 1H), 3.76 (m, 1H), 2.69 (s, 1H),
1.33 (d, J = 6.4 Hz, 3H). Compound 6b was then prepared by the reaction
of 5b (295 mg, 0.538 mmol) with DMTrCl (546 mg, 1.61 mmol) according
to the procedure described for synthesis of 2a. Silica gel chromatography
(1% methanol in dichloromethane containing 0.5% triethylamine) of the
crude product yielded 6b (305 mg, 67% yield, or 44% from 3a) as a yellow
1
foam. H NMR (CDCl3) δ 9.36 (brs, 1H), 8.68 (s, 1H), 8.63–8.61 (m, 3H),
8.25 (s, 1H), 7.94–6.81 (m, 18H), 6.25 (d, J = 2.1 Hz, 1H), 5.68 (m, 1H),
4.86 (s, 2H), 4.34 (m, 1H), 4.29–4.25 (m, 2H), 3.79 (s, 6H), 3.55 (dd, J =
2.7, 10.8 Hz, 1H), 3.44 (dd, J = 4.0, 10.8 Hz, 1H), 2.56 (d, J = 8.0 Hz, 1H),
1.65 (d, J = 6.3 Hz, 3H); HRMS calcd for: C47H45N6O10, [MH+]: 853.3197,
found 853.3200.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-N-phenoxyacetylcytidine
(6c).[19] According to the procedure described for the synthesis of 2a,
compound 6c was prepared by the reaction of 5c (1.24 g, 2.42 mmol)
with DMTrCl (2.46 g, 7.26 mmol) in dry pyridine at rt overnight. Silica
gel chromatography (1% methanol in dichloromethane containing 0.5%
triethylamine) of the crude product yielded 6c[19] (1.67 g, 86% yield) as a
yellow foam. 1H NMR (DMSO-d6) δ 11.03 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H),
8.08 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.76 (m, 1H), 7.57 (m,
1H), 7.40–6.85 (m, 19H), 5.94 (s, 1H), 5.4 (d, J = 7.3 Hz, 1H), 5.21 (dd, J =
15.2, 25.4 Hz, 2H), 4.82 (s, 2H), 4.35 (m, 1H), 4.15 (s, 1H), 4.01 (d, J =
4.8 Hz, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.42–3.36 (m, 2H); HRMS calcd for
C45H43N4O11 [MH+] 815.2928, found 815.2930.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N-phenoxyacetyl-
cytidine (6d). According to the procedure described for the synthesis of
2a, a crude 2ꢁ-O-(α-methyl-o-nitrobenzyl)cytidine (4d) (∼1.42 g) containing
impurities as a yellow foam was prepared from cytidine (0.97 g, 4.0 mmol),
NaH (192 mg, 8.00 mmol) and α-methyl-o-nitrobenzyl bromide (1.43 g,

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
125
6.22 mmol). The molecular weight of 4d was confirmed by MS (ES-API),
calcd for C17H21N4O7 [MH+] 393.1, found 393.2. According to the
procedure described for the synthesis of 5c and 6c, intermediate 4d
(∼1.42 g, 3.60 mmol) was first protected with phenoxyacetyl group to
give a crude 2ꢁ-O-(α-methyl-o-nitrobenzyl)-N 4-phenoxyacetylcytidine (5d)
(1.35 g). The molecular weight of 5d was also confirmed by MS (ES-API),
calcd for C25H27N4O9 [MH+] 527.2, found 527.2. Compound 6d was
then prepared by the reaction of crude 5d (1.35 g) with DMTrCl (2.57 g,
7.58 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1%
methanol in dichloromethane containing 0.5% triethylamine) of the crude
product yielded 6d (459 mg, three steps 14% yield from cytidine) as a
1
yellow foam. H NMR (CDCl3) δ 9.78 (brs, 1H), 8.36 (d, J = 7.5 Hz, 1H),
7.86 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.59 (m, 1H), 7.40–7.20
(m, 14H), 7.09 (d, J = 7.5 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.86 (d, J =
8.5 Hz, 4H), 5.61 (q, J = 6.5 Hz, 1H), 5.56 (s, 1H), 4.65 (s, 2H), 4.35 (m,
1H), 4.11 (m, 1H), 3.92 (m, 1H), 3.81 (s, 6H), 3.62–3.52 (m, 2H), 1.64 (d, J
= 6.5 Hz, 3H); 13C NMR (CDCl3) δ 168.5, 161.8, 158.8, 156.9, 154.8, 148.0,
145.0, 144.2, 139.4, 135.6, 135.3, 133.3, 130.18, 130.15, 129.8, 128.3, 128.1,
127.2, 124.4, 122.4, 114.6, 113.4, 96.4, 88.5, 87.2, 83.4, 82.2, 75.0, 68.2, 67.6,
61.2, 55.3, 23.4; HRMS calcd for C46H44N4O11Na [MNa+] 851.2904, found
851.2874.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)uridine (6e). According to the
procedure described for the synthesis of 2a, compound 6e was prepared
by the reaction of 2ꢁ-O-(o-nitrobenzyl)uridine (5e)[17] (540 mg, 1.42 mmol)
with DMTrCl (1.083 g, 3.20 mmol) in dry pyridine at rt overnight. Silica
gel chromatography (1.5–2% methanol in dichloromethane) of the crude
product yielded 6e (727 mg, 75% yield) as a yellow foam. 1H NMR (CDCl3)
δ 10.05 (brs, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.68
(d, J = 8.0 Hz, 1H), 7.56 (m, 1H), 7.44–7.35 (m, 3H), 7.33–7.25 (m, 7H),
6.83 (d, 4H, J = 8.5 Hz), 6.05 (d, J = 1.8 Hz, 1H), 5.31 (d, J = 8.5 Hz,
1H), 5.28 (d, J = 13.5 Hz, 1H), 5.15 (d, J = 13.5 Hz, 1H), 4.53 (dd, J = 7.5,
5.5 Hz, 1H, 1H), 4.13–4.08 (m, 2H), 3.77 (s, 6H), 3.58 (m, 2H); 13C NMR
(CDCl3) δ 163.8, 158.7, 150.5, 147.9, 144.4, 139.9, 135.3, 135.1, 133.6, 133.0,
130.2, 130.1, 129.8, 128.8, 128.2, 128.0, 127.2, 124.8, 113.3, 102.2, 87.4, 87.1,
83.2, 82.6, 69.4, 68.7, 61.3, 55.3; HRMS calcd for C37H35N3O10Na [MNa+]
704.2220, found 704.2200.
5ꢁ-O-(Dimethoxytrityl)-N2-isobutyryl-2ꢁ-O-(o-nitrobenzyl)guanosine
3ꢁ-
N,N-Diisopropyl(cyanoethyl)phosphoramidite (7a).[20] To a solution of
compound 2a[19] (47 mg, 0.059 mmol) in dry CH2Cl2 (5.0 mL) un-
der Ar, 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) were added. 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 minutes. After the solvent

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Jun Lu et al.
was removed, the crude product was purified by silica gel chromatog-
raphy, eluting with 1% MeOH in CH2Cl2 containing 0.5% Et3N to give
phosphoramidite 7a[20] (56 mg, 95% yield) as a yellow foam. 31P NMR
(CD3CN) δ 152.8, 152.6; HRMS calcd for C51H60N8O11P [MH+]: 991.4119,
found: 991.4110.
5ꢁ-O-(Dimethoxytrityl)-N2-isobutyryl-2ꢁ-O-(α-methyl-o-nitrobenzyl)guan-
osine 3ꢁ-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7b). Phospho-
ramidite 7b was prepared from 5ꢁ-O-(dimethoxytrityl)-N 2-isobutyryl-2ꢁ-
O-(α-methyl-o-nitrobenzyl)guanosine (2b)[19] (255 mg, 0.317 mmol),
N,N -diisopropylethylamine (277 μL, 1.59 mmol), 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite (224 mg, 0.95 mmol), and 1-
methylimidazole (27 μL, 0.32 mmol) as described for 7a. Silica gel
chromatography (1% methanol in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7b (275 mg, 86% yield) as a yellow
foam. 31P NMR (CD3CN) δ 152.7, 152.4; HRMS calcd for C52H62N8O11P
[MH+]: 1005.4276, found: 1005.4276.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-N2-phenoxyacetylguanosine
3ꢁ-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7c). Phosphoramidite 7c
was prepared from 2c (284 mg, 0.332 mmol), N,N -diisopropylethylamine
(290 μL, 1.66 mmol), 2-cyanoethyl N,N -diisopropylchlorophosphoramidite
(234 mg, 0.996 mmol), and 1-methylimidazole (28 μL, 0.33 mmol)
as described for 7a. Silica gel chromatography (1% methanol in
dichloromethane containing 0.5% triethylamine) of the crude product
yielded 7c (270 mg, 77% yield) as a yellow foam. 31P NMR (CD3CN) δ 153.0,
152.7; HRMS calcd for C55H60N8O12P [MH+]: 1055.4069, found: 1055.
4075.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N2-phenoxyacetyl-
guanosine 3ꢁ-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7d). Phos-
phoramidite 7d was prepared from 2d (156 mg, 0.18 mmol),
N,N -diisopropylethylamine (157 μL, 0.90 mmol), 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite (127 mg, 0.54 mmol), and 1-
methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica gel
chromatography (1% methanol in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7d (121 mg, 63% yield) as a yellow
foam. 31P NMR (CD3CN) δ 152.6, 152.5; HRMS calcd for C56H62N8O12P
[MH+]: 1069.4225, found: 1069.4235.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(4,5-dimethoxy-2-nitrobenzyl)-N6-phenoxy-
acetyladenosine 3ꢁ-N,N-diisopropyl (cyanoethyl)phosphoramidite (7e).
Phosphoramidite 7e was prepared from 6a (210 mg, 0.234 mmol),
N,N -diisopropylethylamine (194 μL, 1.11 mmol), 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite (156 mg, 0.66 mmol), and 1-
methylimidazole (18 μL, 0.21 mmol) as described for 7a. Silica gel
chromatography (1% methanol in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7e (225 mg, 88% yield) as a yellow

2ꢁ-O-Photocaged Ribonucleoside Phosphoramidites
127
foam. 31P NMR (CD3CN) δ 152.8, 152.7; HRMS calcd for C57H64N8O13P
[MH+] 1099.4325, found 1099.4309.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N6-phenoxyacetyl-
adenosine 3ꢁ-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7f). Phos-
phoramidite 7f was prepared from 6b (284 mg, 0.33 mmol),
N,N -diisopropylethylamine (276 μL, 1.58 mmol), 2-cyanoethyl
N,N -diisopropylchlorophosphoramidite (221 mg, 0.94 mmol), and
1-methylimidazole (26 μL, 0.30 mmol) as described for 7a. Silica gel
chromatography (1% methanol in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7f (322 mg, 92% yield) as a yellow
foam. 31P NMR (CD3CN) δ 152.6, 152.0; HRMS calcd for C56H62N8O11P
[MH+]: 1053.4276, found: 1053.4277.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-N-phenoxyacetylcytidine
3ꢁ-N,N-Diisopropyl-(cyanoethyl)phosphoramidite
(7g).
Phospho-
ramidite 7g was prepared from 5ꢁ-O-(dimethoxytrityl)-2ꢁ-O-(o-
nitrobenzyl)-N -phenoxyacetylcytidine (6c)[19] (185 mg, 0.23 mmol),
N,N -diisopropylethylamine (192 μL, 1.10 mmol), 2-cyanoethyl
N,N -diisopropylchlorophosphoramidite (154 mg, 0.66 mmol), and
1-methylimidazole (18 μL, 0.21 mmol) as described for 7a. Silica gel
chromatography (0–4% acetone in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7g (215 mg, 92% yield) as a yellow
foam. 31P NMR (CD3CN) δ 152.4, 151.6; HRMS calcd for C54H60N6O12P,
[MH+]: 1015.4007, found: 1015.4011.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(α-methyl-o-nitrobenzyl)-N-phenoxyacetyl-
cytidine 3ꢁ-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7h). Phos-
phoramidite 7h was prepared from 6d (150 mg, 0.18 mmol),
N,N -diisopropylethylamine (158 μL, 0.91 mmol), 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite (129 mg, 0.54 mmol), and 1-
methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica gel
chromatography (0–1% acetone in dichloromethane containing 0.5% tri-
ethylamine) of the crude product yielded 7h (169 mg, 91% yield) as a white
foam. 31P NMR (CD3CN) δ 151.2, 149.4; HRMS calcd for C55H62N6O12P,
[MH+]: 1029.4163, found: 1029.4143.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)uridine
3ꢁ-N,N-Diisopropyl
(cyanoethyl)-phosphoramidite (7i). Phosphoramidite 7i was prepared from
6e (121 mg, 0.18 mmol), N,N -diisopropylethylamine (158 μL, 0.91 mmol),
2-cyanoethyl N,N -diisopropylchlorophosphoramidite (129 mg, 0.54 mmol),
and 1-methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica
gel chromatography (0–2% acetone in dichloromethane containing 0.5%
triethylamine) of the crude product yielded 7i (135 mg, 86% yield) as a white
foam. 31P NMR (CD3CN) δ 150.9, 150.1; HRMS calcd for C46H53N5O11P,
[MH+]: 882.3479, found: 882.3469.
5ꢁ-O-(Dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-N6-benzoyladenosine 3ꢁ-N,N-
Diisopropyl-(cyanoethyl)phosphoramidite (7j).[6,14]
Phosphoramidite

128
Jun Lu et al.
7j was prepared from 5ꢁ-O-(dimethoxytrityl)-2ꢁ-O-(o-nitrobenzyl)-
N 6-benzoyladenosine (6f)[6,14,19]
(99 mg, 0.12 mmol), N,
N -diisopropylethylamine (106 μL, 0.61 mmol), 2-cyanoethyl N,N -
diisopropylchlorophosphoramidite (221 mg, 0.36 mmol), and 1-
methylimidazole (10 μL, 0.12 mmol) as described for 7a. Silica gel
chromatography (1% methanol in dichloromethane containing 0.5%
triethylamine) of the crude product yielded 7j[6,14] (101 mg, 82% yield) as a
yellow foam. 31P NMR (CD3CN) δ 152.9, 152.7.
ACKNOWLEDGMENT
We thank Dr. Qing Dai, Mr. Saurja Dasgupta, Dr. Armando Hernandez,
Mr. Hao Huang and Dr. Sandip Shelke for helpful discussions and critical
comments on the manuscript.
FUNDING
This work was supported by an N.I.H. grant to J.A.P. (1R01-AI081987).
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