7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

Article abstract of DOI:10.1039/c2ob25647a

Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

Full text of DOI:10.1039/c2ob25647a

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PAPER
7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA
major groove†
José M. Ibarra-Soza,‡ Alexi A. Morris,‡ Prasanna Jayalath, Hayden Peacock, Wayne E. Conrad,
Michael B. Donald, Mark J. Kurth and Peter A. Beal*
Received 31st March 2012, Accepted 19th June 2012
DOI: 10.1039/c2ob25647a
Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside
phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog
leads to further structural diversification of the RNA via post-automated RNA synthesis azide–alkyne
cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine
7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi
activity of this type of modification at eight different positions in an siRNA guide strand has been
explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing
specificity and are well-tolerated at specific positions in an siRNA guide strand.
and/or specificity for target RNA without interfering with Ago
binding. This is particularly important in the search for guide
Introduction
Nucleoside analogs incorporated into RNA have a variety of
uses including probing RNA structure and function,1,2 exploring
interactions between RNAs and proteins3 and imparting
favorable properties on small interfering RNAs (siRNAs).4,5
Modifications to the natural structure of siRNAs are known to
improve nuclease resistance,6–8 increase potency9–11 and reduce
off-target effects12 including immune stimulation.13,14 In our pre-
vious studies, we showed that by changing the shape of RNA
nucleobases while maintaining Watson–Crick base pairing one
can generate fully active siRNAs with reduced undesirable
protein binding and reduced immune stimulation.14,15 These
earlier studies focused on modifications to purines that project
substituents into the minor groove of the siRNA duplex.14,15
In this report, we explore an alternative purine modification stra-
tegy where the major groove edge (i.e. the Hoogsteen face) is
modified. Inspection of the published crystal structures of Ago-
RNA–DNA complexes suggest that the major-groove is largely
free of contact with the nuclease of the RNA interference
(RNAi) pathway.16–20 Therefore, the major groove is a logical
location to introduce groups to modulate guide strand affinity
strand modifications that reduce “microRNA-like” off-target
effects that come about from binding to imperfectly matched off-
target mRNAs.21,22 A previous report described the effects of
pyrimidine C5-methyl and C5-propynyl modifications at mul-
tiple positions in the siRNA guide strand.4 In addition, we
reported the effect of N2-alkylated 8-oxo-7,8-dihydro-2′-deoxy-
guanosine analogs at specific positions in the guide strand oppo-
site A in the target, a pairing believed to place the N2 group in
the major groove.23
However, a systematic study focusing on the effect of solitary
major groove modifications at different guide strand positions
has not been reported. Furthermore, for this study we chose to
modify the purine 7-position because, like the C5 of pyrimi-
dines, this site is located in the major groove of duplex structures
and not involved in Watson–Crick base pairing.25 While 7-sub-
stituted 7-deazapurine 2′-deoxyribonucleosides have been used
to modify major groove sites in duplex DNA,26 there are few
examples of effective strategies to introduce these modifications
into duplex RNA nor are there any analyses of their effects
on RNA duplex stability, base pairing specificity or RNAi
activity.27
Here we describe the synthesis of two new phosphoramidites
useful for the modification of the duplex RNA major groove at
adenosines and post-automated synthesis diversification via
azide–alkyne cycloaddition reactions. We also report both the
effects of eight structurally diverse purine 7-position modifi-
cations on duplex RNA stability and pairing specificity as well
as RNAi activity of this type of modification at eight different
positions in an siRNA guide strand.
Department of Chemistry, University of California, Davis, One Shields
Avenue, Davis, California 95616, USA. E-mail: info@chem.ucdavis.edu;
Fax: +1 (530) 752-8995; Tel: +1 (530) 752-8900
†Electronic supplementary information (ESI) available: 1H NMR,
13C NMR, and 31P NMR spectra of all synthesized compounds. See
DOI: 10.1039/c2ob25647a
‡These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2012
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Results and discussion
Synthesis of RNAs containing 7-substituted
8-aza-7-deazaadenosine analogs
We previously reported the synthesis of 7-iodo-8-aza-7-deaza-
adenosine derivative 1 (Scheme 1).28 From this compound,
derivatives 7-propargylamine 2 and 7-ethynyl 3 were obtained in
good yields via Sonogashira couplings29,30 with the requisite
protected alkynes. tert-Butyldimethylsilyl protection at the 2′-
hydroxyls gave 4 and 5, which following phosphitylation at the
3′ positions yielded N,N-diisopropylamino β-cyanoethyl phos-
phoramidites 6 and 7. Both of these phosphoramidites coupled
efficiently during standard automated RNA synthesis yielding
oligoribonucleotides bearing either 7-ethynyl or 7-propargyl-
amine substituted 8-aza-7-deazaadenosine (Fig. 1). The identities
of the resulting RNAs were confirmed by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) analysis,
which verified the removal of protecting groups from the
ethynyl- and propargylamine-modified bases.
Fig. 1 Eight purine 7-position modifications in duplex RNA.
For further structural diversification at the 7-position, we
explored the use of copper-catalyzed azide–alkyne cycloaddition
(click) reactions31–34 with the oligonucleotide bearing 7-ethynyl-
8-aza-7-deazaadenosine. Click products were generated by incu-
bating an aqueous solution of the single-stranded RNA with tris-
[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4-yl)methyl]amine (THPTA)
ligand,35 CuSO4, sodium ascorbate and azide for 6.5 h at
Fig. 2 Azides synthesized for their use in the copper-catalyzed azide–
alkyne cycloaddition with oligonucleotides containing 7-ethynyl-8-aza-
7-deazaadenosine.
ambient temperature,36 with the exception of azide 8 which
required heating to increase solubility. Azides were chosen that
would generate triazoles with a variety of structural features (e.g.
large, small, hydrophilic, charged, neutral and π-stacking). In
particular, triazoles V and VI were chosen for their potential to
simultaneously engage in π-stacking, hydrogen bonding, and
hydrophobic contacts (Fig. 1). Azides 8 and 9 were prepared
from their corresponding bromoketone and bromoalkane,
respectively (Fig. 2).37,38
Each click product was purified from the reaction mixtures by
denaturing polyacrylamide gel electrophoresis (PAGE) and
confirmed by MALDI-MS. Structures for the 8-aza-7-deazaade-
nosine analogs prepared by these approaches, including the
7-ethynyl, 7-propargylamine and the six new triazoles (I–VI),
are shown in Fig. 1.
Effect on RNA duplex stability and base pairing specificity
The effect of the new modifications on duplex stability was
investigated via thermal denaturation (Tm) studies of a 12 base-
pair (bp) RNA duplex (Fig. 1 and 3). The nucleoside analogs
were incorporated into the duplex opposite each of the four
common bases (adenine (A), guanine (G), cytosine (C), and
uracil (U)). Adenine was also incorporated in the same manner
for comparison. Firstly, the Tm of each of the modifications
opposite U was examined to determine the ability of the modifi-
cations to replace A in an A·U pair. The 12 bp duplex containing
an A·U base pair had a measured Tm of 43.1 0.6 °C under
Scheme 1 Synthesis of 7-propargylamine- (6) and 7-ethynyl- (7) phos-
phoramidites: (a) N′-(2-propynyl)-2′′,2′′,2′′-trifluoroacetamide,24 Pd-
(PPh3)4, CuI, Et3N, DMF, rt, 91%. (b) ethynyltrimethylsilane, Pd-
(PPh3)4, CuI, Et3N, DMF, rt, 83%. (c) TBDMSCl, AgNO3, base, THF,
rt, 36% (4), 40% (5). (d) 2-cyanoethyl-(N,N-diisopropylamino)chloro-
phosphite, DIPEA, THF, rt, 70% (6), 84% (7).
6492 | Org. Biomol. Chem., 2012, 10, 6491–6497
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Fig. 3 Thermal denaturation (Tm) of the 12-mer duplexes containing
adenosine and the 7-substituted 8-aza-7-deazaadenosine analogues; pro-
pargylamine, ethynyl, and triazoles I–VI opposite the four natural bases.
Fig. 4 (A) Sequence of siRNA used in this study showing sites of
modification of the guide strand and the structures of the modifications
tested. All siRNAs were prepared with a 5′-phosphorylated guide strand
(p). (B) Knockdown activity of modified siRNAs. Activity is reported
as the ratio of Renilla/firefly luciferase signal at 0.03 nM transfected
siRNA in HeLa cells. Buffer = no added siRNA, unmodified = siRNA
with no modifications.
these conditions. Replacement of A with either the 7-propargyl-
amine analog (Tm of 41.4 1.0 °C) or the piperidine-containing
triazole I (Tm of 41.6 0.3 °C) led to minimal destabilization of
the 12 bp duplex (ΔTm < −2 °C). However, the more sterically
demanding triazoles V and VI were substantially more destabili-
zing (ΔTm > −7 °C for a single modification in this 12 bp
duplex). The 7-ethynyl derivative, along with triazoles II–IV,
was slightly destabilizing relative to adenosine (ΔTm ≈ −3 °C)
(Fig. 3, ESI Table 1†).
As expected for modification of the purine Hoogsteen face,
these alterations had little effect on the base pairing specificity.
Indeed, even the highly destabilizing triazole VI modification
showed selectivity for uracil over the other three nucleotides
(ΔTm ≈ −8 °C comparing match vs. mismatches) that was
similar to that observed for adenine (ΔTm ≈ −10 °C match vs.
mismatches) (Fig. 3, ESI Table 1†).
and 3). For each modified siRNA, we carried out a five-point
concentration profile (0.01, 0.03, 0.1, 1, 10 nM) in the RNAi
assay (ESI Fig. 1†). From these titrations, the concentration of
0.03 nM was chosen for comparison of knockdown activity for
the different modified siRNAs (Fig. 4B). We found that RNAi
activity is altered by these structural changes in a position-
dependent and, at least at one position, a modification-dependent
manner (Fig. 4B). For instance, we found that both modifications
tested were well-tolerated at positions 12 and 20 of the guide
strand. On the other hand, a substantial decrease of knockdown
potency is observed with either modification at positions 1, 3 or
10. These modifications at positions 6 or 18 moderately dimi-
nished potency. Interestingly, at position 15, triazole I is well
tolerated with knockdown indistinguishable from the unmodified
siRNA whereas 7-ethynyl at this location reduces potency. Thus,
triazole I, bearing the N-ethylpiperidine, enhances potency over
the ethynyl precursor, indicating that siRNA potency is sensitive
to the structure of the major groove modification at guide posi-
tion 15. Others have shown that minor groove modifications at
guide position 15 can enhance RNAi activity.40 Together these
results point to the important role of nucleotide structure at this
position and suggest additional modifications here may further
enhance activity. Also, since base pairing to nucleotides 13–16
of the guide has been shown to be a key factor in determining
miRNA-like off-target effects, modulating the interaction
between guide and target in this region is likely to control these
effects.41
Effects on RNA interference
To assess the effect of the C7-modifications on siRNA perform-
ance, we chose for analysis a sequence from the literature
(PIK3CB) with a guide strand rich in adenosines such that
several different positions could be tested with the analogs in
hand (Fig. 4A).39 The target sequence for the PIK3CB siRNA
was inserted into the 3′-UTR of the Renilla luciferase sequence
on the psiCHECK-2 vector as previously described.15 RNAi
activity in HeLa cells was then measured at different siRNA con-
centrations as the ratio of Renilla luciferase activity to control
firefly luciferase encoded on the same plasmid. The siRNA
guide strand was modified with either the 7-ethynyl analog or
triazole I at positions 1, 3, 6, 10, 12, 15, 18 or 20. Triazole I was
chosen for this initial study because of its large size and its
minimal effect on duplex stability compared to adenosine (Fig. 1
This journal is © The Royal Society of Chemistry 2012
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Our observation of decreased potency from either analog at
position 1 is most likely due to binding changes with the Ago2
MID domain. The first nucleotide of the bound guide strand
does not make contact with the target strand.16,17 Other types of
modifications at guide position 1 are also known to decrease
RNAi potency.42–44 The source of decreased potency from
modification of positions 3, 6, 18 and 10 is unknown at this
time, but the latter may be a result of modification of the guide/
target duplex major groove directly across from the cleavage site
of Ago2.17 In addition, the inhibitory effects seen from modifi-
cation of guide position 3 or position 6 mirror Terrazas and
Kool’s observation that C5-propynyl uridine modification at
multiple positions in the 5′ half of the guide strand was detri-
mental to potency.4
were obtained at the University of California, Davis Mass spec-
trometry facility.
4-{[(Dimethylamino)ethylidene]amino}-3-[N′-(2-propynyl)-
2′′,2′′,2′′-trifluoroacetamido]-1-(β-D-ribofuranosyl)-5′-O-(4,4́-
dimethoxytriphenylmethyl)-1H-pyrazolo[3,4-d]pyrimidine (2).
A suspension of 128 (235 mg, 0.307 mmol), Pd(PPh3)4
(36 mg, 0.03 mmol), and CuI (7.6 mg, 0.04 mmol) in anhydrous
DMF (3 mL) was treated with N′-(2-propynyl)-2′′,2′′,2′′-trifluoro-
acetamide24 (227 μL, 1.5 mmol) followed by anhydrous tri-
ethylamine (165 μL, 1.5 mmol). The mixture was stirred under
Ar at room temperature. After the reaction was completed
(TLC), the solvent was removed in vacuo, and the mixture was
partitioned between hexane–EtOAc (3 : 7, 100 mL) and water
(100 mL). The organic layer was dried (Na2SO4), evaporated,
and chromatographed on a flash silica gel column, eluting with
MeOH–DCM (5 : 95) to give 2 (221 mg, 91%) as a white foam.
1H NMR (CDCl3, 600 MHz): δ (ppm) 8.41 (s, 1H), 7.59 (bs,
1H), 7.39–7.15 (m, 9H), 6.80–6.77 (m, 4H), 6.37 (d, J = 6.0,
Hz, 1H), 5.32 (t, J = 3.0, Hz, 1H), 4.92 (t, J = 6.0, Hz, 1H),
4.54 (t, J = 6.0, Hz, 1H), 4.43 (dd, J = 6.0, 18.0 Hz, 1H), 4.37
(dd, J = 6.0, 18.0 Hz, 1H), 4.2 (dd, J = 4.8, 9.6, Hz, 1H), 3.75
(s, 3H), 3.74 (s, 3H), 3.29 (dd, J = 4.8, 12.0 Hz, 1H), 3.22 (dd,
J = 4.8, 12.0 Hz, 1H), 3.18 (s, 3H), 3.13 (s, 3H), 2.22 (s, 3H).
13C NMR (CDCl3, 150 MHz): δ (ppm) 164.4, 160.5, 160.4,
158.6, 157.0, 147.0, 138.4, 138.1, 132.3, 132.1, 130.6, 130.3,
129.8, 128.8, 115.2, 111.0, 90.8, 88.5, 88.1, 85.7, 75.9, 74.0,
66.0, 57.31, 57.30, 32.4, 19.1. ESIHRMS: calcd. for
C40H40F3N7O7 (M + H)+ 788.3020, obsd. 788.3018.
Conclusions
We have synthesized and characterized RNA duplexes modified
with different 7-substituted 8-aza-7-deazaadenosines, including
the six triazoles formed via click reactions from the 7-ethynyl
analog. This type of modification directs substituents into the
duplex RNA major groove. Analogs were identified bearing
large major groove substituents that maintain duplex RNA stabi-
lity and adenosine pairing specificity and are well-tolerated at
specific positions in an siRNA guide strand. In particular, full
RNA interference activity was maintained with two different
modifications at positions 12 and 20 and with triazole I at posi-
tion 15. Since major groove modifications are known to affect
specificity of duplex formation,45 these analogs are interesting
candidates for controlling miRNA-like off-target effects and the
results of such studies will be reported in due course.
4-{[(Dimethylamino)ethylidene]amino}-3-[N′-(2-propynyl)-
2′′,2′′,2′′-trifluoroacetamido]-1-(β-D-ribofuranosyl)-5′-O-(4,4′-
dimethoxytriphenylmethyl)-2′-O-(tert-butyldimethylsilyl)-1H-
pyrazolo[3,4-d]pyrimidine (4). To a stirred solution of 2
(217 mg, 0.275 mmol) and tert-butylchlorodimethylsilane
(53.9 mg, 0.358 mmol) in freshly distilled THF (4 mL) was
added AgNO3 (62.8 mg, 0.37 mmol) followed by DIEA
(174 μL, 1 mmol) and stirring was continued at room tempera-
ture for 12 h. The reaction was then diluted with EtOAc
(25 mL), filtered, and washed with 5% aqueous NaHCO3 (1 ×
30 mL). The organic portion was dried (Na2SO4), filtered and
concentrated under reduced pressure. Purification by flash
column chromatography on silica gel with EtOAc–hexane
(30 : 70) as eluent gave 4 (87 mg, 36%). 1H NMR (CD2Cl2,
600 MHz): δ (ppm) 8.50 (s, 1H), 7.45–7.43 (m, 2H), 7.36–7.34
(m, 4H), 7.24–7.21 (m, 2H), 7.18–7.15 (m, 1H), 7.07 (bs, 1H),
6.82–6.77 (m, 4H), 6.32 (d, J = 6.0, Hz, 1H), 5.30 (t, J =
3.0 Hz, 1H), 5.05 (t, J = 6.0 Hz, 1H), 4.41 (dd, J = 6.0, 18 Hz,
1H), 4.35 (dd, J = 6.0, 18 Hz, 1H), 4.28 (dd, J = 6.0, 12 Hz,
1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.31 (dd, J = 6.0, 12 Hz, 1H),
3.20 (bs, 3H), 3.17 (dd, J = 6.0, 12 Hz, 1H), 3.14 (bs, 3H), 2.66
(d, J = 6.0 Hz, 1H), 2.22 (s, 3H), 0.81 (s, 9H), −0.01 (s, 3H),
−0.16 (s, 3H). 13C NMR (150 MHz, CD2Cl2) δ 167.6, 163.8,
163.7, 162.2, 150.4, 141.8, 141.4, 135.7, 135.4, 133.8, 133.7,
133.1, 132.0, 118.5, 114.9, 93.8, 91.5, 91.4, 89.4, 80.3, 77.3,
69.4, 60.6, 59.2, 59.0, 58.8, 58.7, 58.5, 35.7, 30.8, 23.2, 22.3,
0.2, 0.0. The identity of 2′-O-TBDMS isomer (vs. 3′-O-TBDMS
isomer) was confirmed by 2D-NMR (COSY). ESIHRMS: calcd.
for C46H54F3N7O7Si (M + H)+ 902.3884, obsd. 902.3862.
Experimental
General synthetic procedures
Glassware for all reactions was oven dried at 175 °C overnight
and cooled in a desiccator prior to use. Reactions were carried
out under an atmosphere of dry argon when anhydrous con-
ditions were necessary. All reagents were purchased from com-
mercial sources (Sigma/Aldrich or Fischer Scientific) and were
used without further purification unless noted otherwise. Liquid
reagents are introduced by oven-dried microsyringes. Tetrahydro-
furan was dried in a solvent purification system that passes
solvents through two columns of dry neutral alumina. Thin layer
chromatography (TLC) was performed with Merck silica gel 60
F254 precoated TLC plates. Short and long wave visualization
was performed with a Mineralight multiband ultraviolet lamp at
254 and 365 nm, respectively. Flash column chromatography
was performed with Merck silica gel (Sorbent technologies,
60–200 mesh). Radial chromatography was preformed with
1
Merck silica gel 60 PF 254 containing CaSO4. H, 13C, and 31P
Nuclear Magnetic Resonance spectra of pure compounds were
acquired with Varian VNMRs 600 and Mercury 300 spec-
trometers. Chemical shifts are reported in parts per million
(ppm) in reference to a solvent peak. The abbreviations s, t, m,
bs, dd, d stand for singlet, triplet, multiplet, broad singlet,
doublet of doublets and doublet. High-resolution mass spectra
6494 | Org. Biomol. Chem., 2012, 10, 6491–6497
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4-{[(Dimethylamino)ethylidene]amino}-3-[N′-(2-propynyl)-
2′′,2′′,2′′-trifluoroacetamido]-1-(β-D-ribofuranosyl)-5′-O-(4,4
4H), 6.32 (d, J = 4.7 Hz, 1H), 5.11 (t, J = 5.0 Hz, 1H), 4.28 (q,
J = 4.7 Hz, 1H), 4.14 (dd, J = 8.2, 4.5 Hz, 1H), 3.76 (s, 3H),
3.75 (s, 3H), 3.39–3.16 (m, 8H), 2.67 (d, J = 5.1 Hz, 1H), 2.20
(s, 3H), 0.84 (s, 9H), 0.23 (s, 9H), 0.01 (s, 3H), −0.13 (s, 3H).
13C NMR (CD2Cl2, 75 MHz): δ (ppm) 163.4, 162.0, 159.1,
159.0, 157.2, 155.9, 145.8, 136.6, 136.5, 130.8, 130.7, 129.9,
128.7, 128.3, 127.0, 113.6, 109.6, 98.9, 97.3, 89.2, 86.7, 84.4,
75.2, 72.4, 64.6, 55.7, 39.0, 38.8, 26.0, 18.4, 17.5, 0.0, −4.6,
−4.9. The identity of 2′-O-TBDMS isomer (vs. 3′-O-TBDMS
isomer) was confirmed by 2D-NMR (COSY). ESIHRMS: calcd
for C46H61N6O6Si2 (M + H)+ 849.4191, obsd 849.4201.
́
-
dimethoxytriphenylmethyl)-3′-O-[(2-cyanoethyl)(N,N-diisopropyl-
amino)phosphino]-2′-O-(tert-butyldimethylsilyl)-1H-pyrazolo-
[3,4-d]pyrimidine (6). N,N-Diisopropylethylamine (87 μL,
0.5 mmol), and 2-cyanoethyl-(N,N-diisopropylamino)chloro-
phosphite (103 μL, 0.435 mmol) were consecutively added to a
solution of 4 (157 mg, 0.174 mmol) in freshly distilled THF
(1 mL). The resulting reaction mixture was stirred at room temp-
erature for 2 h. It was then diluted with EtOAc (30 mL), filtered,
and washed with 5% (w/v) aqueous NaHCO3 (2 × 15 mL). The
organic portion was dried (Na2SO4), filtered and concentrated
under reduced pressure. Purification was performed by radial
chromatography (1 mm plate) on silica gel using EtOAc–hexane
(40 : 60) as eluent to give 6 (134 mg, 70%). 31P NMR (CD2Cl2,
121 MHz): δ (ppm) 151.24, 150.78. ESIHRMS: calcd. for
C55H71N8O8PSi (M + H)+ 1102.4963, obsd. 1102.4956.
4-{[(Dimethylamino)ethylidene]amino}-3-[(trimethylsilyl)ethynyl]-
1-(β-D-ribofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)-3′-
O-[(2-cyanoethyl)(N,N-diisopropylamino)phosphino]-2′-O-(tert-
butyldimethylsilyl)-1H-pyrazolo[3,4-d]pyrimidine (7). N,N-Di-
isopropylethylamine (105 μL, 0.60 mmol) and 2-cyanoethyl-
(N,N-diisopropylamino)chlorophosphite (45 μL, 0.20 mmol)
were consecutively added to a solution of 5 (80 mg, 0.10 mmol)
in anhydrous DCM (1 mL). The resulting reaction mixture was
stirred under Ar at room temperature for 10 h. It was then diluted
with EtOAc (30 mL) and washed with sat. NaHCO3 (15 mL).
The organic portion was dried with Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was chromato-
graphed on a flash silica column, eluting with EtOAc–hexane
(80 : 20) to give 7 (88 mg, 84%) as a white foam. 31P NMR
(CD2Cl2, 121 MHz): δ (ppm) 151.11, 149.67. ESIHRMS: calcd.
for C55H78N8O7PSi2 (M + H)+ 1049.5270, obsd. 1049.5290.
4-{[(Dimethylamino)ethylidene]amino}-3-[(trimethylsilyl)ethynyl]-
1-(β-D-ribofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)-1H-
pyrazolo[3,4-d]pyrimidine (3). A suspension of 128 (1.26 g,
1.64 mmol), Pd(PPh3)4 (0.48 g, 0.41 mmol), and CuI (0.08 g,
0.43 mmol) in anhydrous DMF (16 mL) was treated with ethy-
nyltrimethylsilane (1.14 mL, 8.20 mmol) followed by anhydrous
triethylamine (1.14 mL, 8.20 mmol). The mixture was stirred
under Ar at room temperature. After the reaction was completed,
the solvent was removed in vacuo, and the residue was
chromatographed on a flash silica column, eluting with MeOH–
DCM (0 : 100 → 3 : 97 → 5 : 95) to give 3 (1.01 g, 83%) as a
foam. The progress of the reaction was monitored by following
the disappearance of 1 with the use of a Q-Trap ESI-Mass spec-
N-(5-(2-Azidoacetyl)-4-methylthiazol-2-yl)benzamide (8). N-
(5-(2-Bromoacetyl)-4-methylthiazol-2-yl)benzamide38 (50 mg,
0.148 mmol) was stirred with sodium azide (10 mg,
0.163 mmol) at room temperature in DMF (1 mL) for 3 hours.
Then 10 mL of ice-cold water was added and the resulting pre-
cipitate was filtered. The off-white solid was purified by flash
chromatography with EtOAc–hexane (1 : 1) yielding 8 as a white
solid (37 mg, 85%). mp decomposition at 132 °C. IR (neat) νmax
3195, 2119, 1684, 1666, 1541, 1497, 1372, 1319, 1292, 1221,
1
trometer. H NMR (CD2Cl2, 300 MHz): δ (ppm) 8.37 (s, 1H),
7.44–7.41 (m, 2H), 7.31–7.13 (m, 7H), 6.78 (d, J = 2.4 Hz, 2H),
6.75 (d, J = 2.4 Hz, 2H) 6.38 (d, J = 3.9 Hz, 1H), 4.93 (t, J =
4.5 Hz, 1H), 4.51 (t, J = 5.1 Hz, 1H), 4.21 (dd, J = 9.1, 5.0 Hz,
1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.35–3.20 (m, 5H), 3.12 (s, 3H),
2.12 (s, 3H), 0.26 (s, 9H). 13C NMR (CD2Cl2, 75 MHz):
δ (ppm) 163.2, 162.0, 159.02, 158.98, 156.7, 155.2, 145.8,
136.48, 136.47, 130.64, 130.55, 129.9, 128.6, 128.3, 127.0,
113.5, 109.3, 99.2, 97.2, 89.5, 86.6, 84.1, 74.0, 72.2, 64.7, 55.7,
39.1, 38.9, 31.2, 17.6, 0.0. ESIHRMS: calcd. for C40H47N6O6Si
(M + H)+ 735.3326, obsd. 735.3336.
1
910, 875 cm−1. H NMR (CDCl3, 600 MHz): δ (ppm) 7.92 (d,
J = 7.8 Hz, 2H), 7.64 (t, J = 7.2 Hz, 1H), 7.51 (t, J = 7.2 Hz,
2H), 4.30 (s, 2H), 2.35 (s, 3H). 13C NMR (CDCl3, 150 MHz): δ
(ppm) 186.7, 165.7, 160.9, 157.9, 133.8, 131.5, 129.4, 128.0,
121.5, 57.1, 18.3. ESIHRMS calcd. for C13H11N5O2S (M + H)+
301.0633, obsd. 301.0713.
4-{[(Dimethylamino)ethylidene]amino}-3-[(trimethylsilyl)ethynyl]-
1-(β-D-ribofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-
O-(tert-butyldimethylsilyl)-1H-pyrazolo[3,4-d]pyrimidine (5).
Triethylamine (151 μL, 1.08 mmol) and tert-butylchloro-
dimethylsilane (0.10 g, 0.63 mmol) were consecutively added to a
solution of 3 (437 mg, 0.57 mmol) in anhydrous THF (10 mL).
AgNO3 (0.11 g, 0.63 mmol) was added after stirring for 5 min.
The resulting mixture was stirred under Ar at room temperature
for 19 h. It was then diluted with EtOAc (25 mL), filtered, and
washed with sat. NaHCO3 (1 × 30 mL). The organic portion was
dried (Na2SO4), filtered, and concentrated under reduced
pressure. Purification was carried out by flash column chromato-
graphy on silica gel, eluting with EtOAc–hexane (80 : 20) to
give 5 (198 mg, 40%) as a yellow foam. 1H NMR (CD2Cl2,
300 MHz): δ (ppm) 8.52 (s, 1H), 7.50–7.47 (m, 2H), 7.38–7.31
(m, 4H), 7.29–7.23 (m, 2H), 7.20–7.14 (m, 1H), 6.82–6.76 (m,
( )-2-(1-Azidopropan-2-yl)-1-((3-isopropylisoxazol-5-yl)methyl)-
1H-indazol-3(2H)-one (9). ( )-2-(1-Bromopropan-2-yl)-1-((3-iso-
propylisoxazol-5-yl)methyl)-1H-indazol-3(2H)-one37 (149 mg,
394 mmol) was added to a 5–10 mL microwave vial and dis-
solved in DMF (2.0 mL). Sodium azide (30.7 mg, 473 mmol)
was added and the vial was sealed and placed in an oil bath at
60 °C for 3 hours. The DMF was then removed under reduced
pressure and the crude material was dissolved in EtOAc
(30 mL). This solution was then washed with water (30 mL) and
brine (30 mL), dried over sodium sulfate, and concentrated. This
crude material was purified by flash chromatography with
EtOAc–hexane (40 : 60) to afford 9 as a pale yellow solid
(133 mg, 99%). mp 86–87 °C. IR (neat) νmax 2969, 2930, 2904,
2876, 2091, 1675, 1607, 1461, 1334, 1312, 1251, 1235 cm−1
.
1H NMR (CDCl3, 600 MHz): δ (ppm) 7.73 (d, J = 7.8 Hz, 1H),
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6491–6497 | 6495

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7.52 (ddd, J = 7.5, 7.5, 1.0 Hz, 1H), 7.22–7.14 (m, 2H), 5.57 (s,
1H), 4.88 (d, J = 16.9 Hz, 1H), 4.82 (d, J = 16.9 Hz, 1H),
4.21–4.14 (m, 1H), 4.03 (dd, J = 12.5, 8.7 Hz, 1H), 3.51 (dd,
J = 12.5, 5.5 Hz, 1H), 2.82 (m, J = 6.9 Hz, 1H), 1.42 (d, J =
7.0 Hz, 3H), 1.08 (d, J = 6.9 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H).
13C NMR (CDCl3, 150 MHz): δ (ppm) 169.3, 166.4, 164.5,
150.9, 133.1, 124.2, 123.6, 120.2, 112.6, 101.4, 53.5, 53.4, 46.7,
26.5, 21.5, 21.5, 15.8. ESIHRMS calcd. for C17H20N6O2
(M + H)+ 341.1648, obsd. 341.1722.
the triazole-containing RNAs corresponding to the clicked
product. The click reactions performed on the crude ethynyl-
containing RNA exhibited comparable efficiency and gel-shift
patterns to the pure reactions with ethynyl-containing RNA,
though bands corresponding to by-products of incomplete coup-
ling were also visible. MALDI-TOF analysis confirmed identity
of the click products (as described above).
List of mass values, [M + H]+, for the triazole-containing
RNAs: Triazole I: Calc. 4054.6, Obs. 4054.5; Triazole II: Calc.
4162.6, Obs. 4162.6; Triazole III: Calc. 4001.5, Obs. 4001.1;
Triazole IV: Calc. 4118.7, Obs. 4118.5; Triazole V: Calc.
4240.8, Obs. 4042.8; Triazole VI: Calc. 4201.7, Obs. 4201.6.
Biochemical procedures
Synthesis, purification and quantification of RNA 12-mer.
RNA oligonucleotides were synthesized on an ABI 394 syn-
thesizer (DNA/Peptide Core Facility, University of Utah, Salt Lake
City) using 5′-DMTr protected β-cyanoethyl phosphoramidites
on a 1.0 mmol scale with coupling times of 25 min for increased
coupling efficiency of amidites 6 and 7. All RNAs were depro-
tected as previously described.46 The RNA oligonucleotides
containing ethynyl and propargylamine modifications were gel-
purified and quantified as previously described.14 The identity of
the RNAs was confirmed by MALDI mass spectrometry.
Thermal melting (Tm) analysis. The thermal stability of the
ethynyl, propargylamine and triazole-containing RNAs were
analyzed in a 12 bp duplex of sequence derived from human
glutamate receptor B subunit pre-mRNA using the following
buffer conditions: 10 mM Tris-HCl, pH 7.8, 0.1 mM EDTA and
100 mM NaCl.36 The values reported in ESI Table 1† are an
average of three denaturation experiments, with the experimental
temperature range noted for each RNA duplex. The error bars in
the graph (Fig. 3) indicate standard deviation.
List of mass values, [M + H]+, for the RNA containing pro-
pargylamine and ethynyl modifications: Propargylamine: Calc.
3929.5, Obs. 3929.5; Ethynyl: Calc. 3900.4, Obs. 3900.5.
Synthesis, purification and quantification of 21-mer RNA.
RNA oligonucleotides were synthesized as described above.
5′ Phosphorylation of siRNA guide strands was accomplished
using the chemical phosphorylation reagent (Glenn Research
Corporation). RNAs were deprotected, gel-purified and analyzed
by MALDI-MS, as described above.
Mass spectrometry analysis of RNA 12-mer. Mass spectra
were obtained on an Applied Biosystems 4700 MALDI-TOF
mass spectrometer operating in linear mode. Desalted samples
(2 μL of 10 μM) were combined with an equal volume of matrix
(either a 10 : 1 mixture of 50 mg mL−1 solution of 3-hydroxy-
picolinic acid in 1 : 1 acetonitrile–H2O and a 100 mg mL−1 solu-
tion of di-ammonium hydrogen citrate in H2O, or a saturated
solution of 6-aza-2-thiothymine in 0.1 M aqueous dibasic
ammonium citrate), and no more than 1 μL was applied to the
target and air-dried. Mass spectra were recorded in the positive
ionization mode and calibrated to an internal DNA standard of
4366.8 Daltons.
List of mass values, [M + H]+, for all ethynyl-modified
siRNAs: (G = guide, indicates position of modification, calcu-
lated mass is the same for all siRNAs modified in this way)
Calc: 6851.1, Observed: G1 = 6849.3; G3 = 6849.3; G6 =
6849.0; G10 = 6849.9; G12 = 6850.0; G15 = 6849.9; G18 =
6851.1; G20 = 6849.8.
Click reactions on siRNA guide strands. Reaction and purifi-
cation of the ethynyl-modified siRNAs with Triazole I were exe-
cuted in the same fashion as above with 15 nmol of crude RNA
for each reaction. MALDI-MS analysis confirmed the identity of
the click products.
Click reactions on 12-mer RNAs. A dry pellet of 10–20 nmol
pure (purified as described above) or 40–60 nmol crude ethynyl-
containing RNA was dissolved in 1 μL of H2O, then treated
sequentially with tris-[1-(3-hydroxypropyl)-1H-[1,2,3]triazol-4-
yl)methyl]amine (THPTA) ligand35 (1 μL, 1 M in H2O), CuSO4
(1 μL, 100 mM in H2O), sodium ascorbate (1 μL, 1 M in H2O)
and 1 μL of the corresponding azide. Azides were prepared at
the following concentrations: 1-(2-azidoethyl)-piperidine,47
50 mM in 0.5 M Tris-HCl, pH 8.0; N-azidoacetyl-D-mannos-
amine,48 0.5 M in H2O; azides 8 and 9, 150 mM in DMSO;
3-azido-1-propanol and 11-azido-3,6,9-trioxaundecan-1-amine,
150 mM in H2O. The resulting reaction solutions were incubated
at room temperature for 6.5 h except for the click reaction with
azide 8, to which 1 μL more of DMSO was added and incubated
at 35 °C to improve azide solubility. The reaction mixtures were
diluted to 2× the original volume with PAGE loading buffer
(80% formamide containing 10 mM EDTA). The 12-mer RNA
was gel purified and quantified as above. Lyophilization gave
white pellets, which were fully soluble in H2O. In the case of
pure ethynyl-containing RNA, a single band migrated faster than
List of mass values, [M + H]+, for all Triazole I-modified
siRNAs: (G = guide, indicates position of modification, calcu-
lated mass is the same for all siRNAs modified in this way)
Calc: 7005.3, Observed: G1 = 7007.4; G3 = 7005.9; G6 =
7004.2; G10 = 7002.5; G12 = 7006.4; G15 = 7006.4; G18 =
7003.6; G20 = 7003.6.
siRNA duplex formation. siRNA duplex hybridization was
accomplished by combining equal amounts of purified passenger
and modified guide strands to a final concentration of 5 μM in
10 mM Tris-HCl, 50 mM KCl, pH 7.5. The samples were heated
at 95 °C for 5 minutes followed by slow-cooling to room tem-
perature over a period of approximately 2 h.
Cell culture. HeLa cells (ATCC) were grown at 37 °C in
humidified 5% CO2 in Dulbecco’s modified Eagle’s medium
(DMEM, GIBCO) supplemented with 10% fetal bovine serum
(FBS, GIBCO) and 100 Units per mL−1 penicillin and 100 μg mL−1
streptomycin (GIBCO, 1× Pen Strep). The cells were maintained
in exponential growth.
6496 | Org. Biomol. Chem., 2012, 10, 6491–6497
This journal is © The Royal Society of Chemistry 2012

View Article Online
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Transfection and RNAi activity assay. HeLa cells were
reverse-transfected using siPORT NeoFX transfection reagent
(Ambion) according to the manufacturer’s instructions. Cells
were grown in flasks to approximately 80–90% confluence then
trypsinized (0.025% trypsin-EDTA, GIBCO) and diluted in fresh
medium (DMEM, 10% FBS, 1× Pen Strep) to a concentration of
1 × 105 cells per mL−1. The psiCHECK-2 plasmid (Promega),
containing the reporter genes Renilla and firefly luciferase
(hRluc and hluc+, respectively), was used as the vector. The
PIK3CB siRNA target sequence was inserted in the 3′ UTR of
the Renilla luciferase gene (psiCHECK-2-PIK3CB), between
the NotI and XhoI restriction sites (see below), allowing this
luciferase to be used as a reporter of siRNA potency, while the
firefly luciferase was used as an internal control. Plasmid and
siRNA cotransfections and 96 well plate assay were performed
as previously described,14 with the exception that 20 ng per well
of psiCHECK-2-PIK3CB were used for these assays. For the
data in Fig. 4, three separate assays were executed where all
modifications were tested in the same 96 well plate, then aver-
aged to give the values and standard deviations plotted.
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Sequence inserted between Xho1 and Not1 in psiCHECK-
2-PIK3CB plasmid: 5′-GCACATCTCCTAAUATGAATCCTAT-
CAGAA-3′.
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Acknowledgements
P.A.B. acknowledges the National Institutes of Health for
financial support in the form of grant R01GM080784. The
authors would like to acknowledge Erik Q. Fostvedt for helpful
scientific discussions. J.M.I. thanks the Alfred P. Sloan Minority
Ph.D. Program for fellowship support.
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