Regiospecific solid-phase synthesis of branched oligoribonucleotides that mimic intronic lariat RNA intermediates

Article abstract of DOI:10.1021/jo4024182

We have developed new solid phase methods for the synthesis of branched RNAs that mimic intronic lariat RNA intermediates. These methods produce branched oligoribonucleotide sequences of arbitrary length, base composition, and regiochemistry at the branch

Full text of DOI:10.1021/jo4024182

Article
pubs.acs.org/joc
Regiospecific Solid-Phase Synthesis of Branched
Oligoribonucleotides That Mimic Intronic Lariat RNA Intermediates
Adam Katolik,† Richard Johnsson,† Eric Montemayor,‡ Jeremy G. Lackey,† P. John Hart,‡,§
and Masad J. Damha*,†
†McGill University, 801 Sherbrooke Street West, Montrea
́ ́
l, Quebec H3A 0B8, Canada
‡University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229, United States
§Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, Texas 78229, United States
S
* Supporting Information
ABSTRACT: We have developed new solid phase methods for
the synthesis of branched RNAs that mimic intronic lariat RNA
intermediates. These methods produce branched oligoribonucleo-
tide sequences of arbitrary length, base composition, and
regiochemistry at the branchpoint junction. The methods utilize
branching monomers that allow for the growth of each branch
regioselectively from any of the hydroxyl positions (5′, 3′, or 2′) at
the branch-point junction. The integrity and branchpoint
connectivity of the synthetic products have been confirmed by
HPLC and MS analysis, and cleavage of the 2′,5′ linkage by
recombinant debranching enzyme. Nonhydrolyzable branched
RNA analogues containing arabinose instead of ribose at the
branchpoint junction were shown to inhibit debranching activity
and, hence, represent “decoys” for sequestering RNA binding
proteins thought to drive amyotrophic lateral sclerosis (ALS).
also recently yielded high-resolution crystal structures of bRNA
bound to the Dbr1 active site (Montemayor et al., manuscript
in preparation), establishing a platform for rationally designing
new and more optimal inhibitors of the enzyme. Toward this
end, the synthetic protocols described here allow production of
milligram quantities of branched substrates needed for further
structural and pharmacological investigation.
Ogilvie and co-workers have prepared branched tri- and
tetranucleotides containing identical nucleotides at the
branchpoint 2′ and 3′ positions, and their methods have
subsequently been adapted to the synthesis of bRNA isomers
with different nucleotides at the 2′ and 3′ positions.18
Regiospecific synthesis of tetranucleotide branchpoints have
been performed by Kierzek et al. using solution-phase
phosphoramidite chemistry.19 Chattopadhyaya and co-workers
have synthesized and structurally characterized heptameric
bRNAs and RNA minilariats with self-cleavage properties.20,21
Although all of these approaches have their own advantages,
they are generally labor intensive, particularly in the synthesis of
medium size bRNAs (>5 nucleotides). As a result, much
current effort has focused on ways to synthesize larger bRNAs
via solid-phase methods. Our first such approach utilized 2′,3′-
bis-O-phosphoramidite to couple two support-bound RNA
INTRODUCTION
■
The spliceosome removes introns from eukaryotic precursor
mRNA (pre-mRNA) in the form of RNA lariats that possess
unusual 2′,5′-phosphodiester linkages (Figure 1).1,2 This
linkage endows enormous stability to the RNA lariat3 and
must be hydrolyzed by the RNA intron debranching enzyme
(Dbr1)4 before the lariat can be efficiently metabolized or
converted into other critical cellular factors such as snoRNA5 or
miRNA.6 Dbr1 has also been implicated in the propagation of
retrotransposons7 and in the replication of HIV-1 in human
cells, ostensibly through a 2′,5′-phosphodiester intermediate
that is transiently formed during reverse transcription.8
However, this claim has since been challenged by other
groups.9,10 Several other branched structures such as multicopy
single-stranded DNAs (msDNA) have been detected in living
cells,11,12 all of which contain the same common structural
features of lariat RNA, namely, vicinal 2′,5′- and 3′,5′-
phosphodiester bonds at a branchpoint nucleotide (Figure 1).
Synthetic branched RNAs (bRNAs) are important model
systems for studying bRNA structure,13 pre-mRNA splicing14
and intron debranching.15,16 They also represent lead
compounds in the development novel nucleic acid-based
therapeutics.17 For example, inhibition of Dbr1 causes RNA
lariats to accumulate in the cytoplasm and act as “decoys” in
sequestering toxic RNA binding proteins thought to drive
amyotrophic lateral sclerosis (ALS).17 Structural studies have
Received: October 30, 2013
Published: January 8, 2014
© 2014 American Chemical Society
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Figure 1. Structures of lariat and branched RNA (bRNA). The 2′,5′-phosphodiester bond found in these RNAs (blue) is hydrolyzed by the RNA
intron debranching enzyme Dbr1. After hydrolysis, the phosphate moiety remains attached to the 5′ position of guanosine, and the branchpoint
adenosine has a 2′ hydroxyl group.
chains, thus forming a branch juncture with the desired vicinal
2′,5′- and 3′,5′-phosphodiester bonds.22 With this method,
dendritic (multibranched), Y-shaped (branched), and V-shaped
(branch lacking the 5′ branch segment) molecules having
identical 2′ and 3′ chains were readily assembled.22,23 The
method was later adapted to the synthesis of bRNA mixtures
with 2′ and 3′ chains of different base composition.24
RNA synthesis methods.33 Ready availability of the forward and
reverse RNA phosphoramidite monomers and trivial synthesis
of the branchpoint monomers allows this approach to be used
in synthesizing milligram (micromol) quantities of bRNA of
arbitrary length and base composition around the branch-point
junction, limited only by the total length of the oligonucleo-
tides. Additionally, we describe a third regioselective approach
to synthesize branched compounds whose branchpoint sugar
moiety has been modified to an arabinose.
Sproat and colleagues have reported novel methods toward
the regiospecific solid-phase synthesis of small to medium-sized
bRNA molecules25,26 and branched DNA/RNA chimeras.27
Although elegant, their methodologies necessitated numerous
orthogonal protecting groups as well as in-house synthesis of
complex sets of phosphoramidite building blocks and
branchpoint introduction synthons, making this approach also
highly labor intensive. Similarly, a divergent approach has been
adapted to the regioselective synthesis of branched DNA28−30
and branched DNA/RNA chimeras similar to the msDNA
molecule of the prokaryote Myxococcus xanthus.31 The latter
work relied on the chemoselective removal of the 2′-TBDMS
group (embedded within a 2′-fpmp protected RNA) to allow
chain extension from the 2′-hydroxyl of the branch with
inverted DNA 5′-phosphoramidites. As reverse RNA phosphor-
amidite monomers were not available at the time, the strategy
was initially incapable of generating all-RNA branched
structures. Furthermore, extra care was necessary to prevent
cleavage of the oligomer from the CPG solid support during
fluoride-mediated cleavage of the TBDMS group. Silverman
and colleagues have introduced an elegant, deoxyribozyme-
based system that catalyzes the formation of 2′,5′-branched
RNA linkages with very high selectivity.32 In contrast to
synthetic methods, this approach is limited by synthesis scale
(pico/nanomol), a requirement of a deoxyribozyme (>70
nucleotides), limited sequence tolerance at the branchpoint
nucleotide, and variable binding domain sequence of the excess
DNA “catalyst” used.
RESULTS AND DISCUSSION
■
The present work is an extension of our previously described31
“divergent” method for the synthesis of chimeric branched
DNA-RNA and focuses on the synthesis of branched nucleic
acids consisting entirely of ribonucleotide units (see Scheme
2A,B). A key development of the present work is the use of
branch-site 2′-acetal levulinic ester (ALE) (A) and 2′-
dimethoxytrityl (DMTr) (B) protected monomers that are
used at the branchpoint and their use in conjunction with the
reverse (R) and standard (S) 2′-TBDMS monomers. A suitable
protecting group for the 2′-hydroxyl of the branchpoint must
be orthogonal to protecting groups on the sugar (5′ and 3′
hydroxyls) and the nucleobases (exocyclic amines). It must
additionally be orthogonal to the TBDMS protecting group at
the 2′ hydroxyls of all remaining nucleotides, so that only the 2′
hydroxyl of the designated branchpoint can be deprotected
selectively. The ALE and DMTr groups meet this requirement
as they can be removed by hydrazinolysis or acidolysis,
respectively, and these conditions do not affect the 2′-
TBDMS or amide-based N-protecting groups generally used
for RNA synthesis.
Synthesis of Branchpoint Monomers. 3′,5′-Bis-silylated
N-Bz adenosine with a thiomethyl ether in position C2′ (1)34
was activated with SO2Cl2 and reacted with cesium bicarbonate
activated levulinic acid to yield the acetal levulinic ester (ALE)
protected compound 2 in 59% yield.35 The ALE group35 is
used instead of levulinic ester to avoid migration of the ester
from 2′ to 3′. Subsequent cleavage of the silyl protection
yielded compound 3 in 73% that was tritylated and
phosphitylated to give compound A (Scheme 1A). Our lab
has previously developed 2′-DMTr protected guanosine
phosphoramidites for the synthesis of oligoribonucleotides
Here, we describe reliable procedures for large-scale,
regiospecific solid-phase synthesis of bRNAs with mixed base
compositions. Our method utilizes branch-site monomers that
allow for the growth of each branch regioselectively from any of
the hydroxyl positions (5′, 3′, or 2′), providing an end product
of TBDMS-protected bRNA molecules that can be released
from the support and deprotected following well established
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Scheme 1. Synthesis of Branchpoint Synthons
with a 2′ phosphate monoester moiety.36 Compound 6 was
protected with DMTr in position C2′ followed by silyl
deprotection with triethylamine trihydrofluoride (TREAT-
HF) to give compound 7 in 55% yield. Compound 7 was
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Scheme 2. Solid-Phase Synthesis of Branched RNAs
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Scheme 2. continued
then treated with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl-
uronium tetrafluoroborate (TBTU) and levulinic acid to
selectively install the levulinyl group on the 5′-hydroxyl in
50%. Finally, phosphitylation of the 3′-hydroxyl group afforded
branching monomer B in 56% yield (Scheme 1B). The 5′,3′
silyl protected N-Bz arabinoadenosine (10) was reacted with
levulinic anhydride in the presence of N,N-dimethylaminopyr-
idine (DMAP) to yield compound 11 in 74% yield. Subsequent
cleavage of the silyl protection yielded compound 12 in 75%
that was tritylated and phosphitylated to give compound Z
(Scheme 1C). NMR characterization data (spectra) are
provided in the Supporting Information.
Synthesis of Native Branched RNAs by Method A. This
method synthesizes the 2′ branched segment from an
adenosine unit located in the middle of the RNA chain. The
key features of this method are (a) solid-supported synthesis of
an RNA strand (via the incorporation of standard TBDMS
RNA monomers (S)) bearing a single 2′-ALE unit at the
branch-site monomer (A); (b) selective sequential removal of
cyanoethyl phosphodiester groups followed by the 2′-ALE
group; and (c) 5′ to 3′ growth of the 2′-branch segment using
reverse RNA monomers (R) (Scheme 2A). Specifically, the first
two branch segments are synthesized in the conventional 3′ to
5′ direction using commercially available 2′-TBDMS 3′-
phosphoramidite monomers followed by branching synthon
A, thus yielding a linear RNA chain (5′-NN′...N2′N1′-A2′‑ALE-
NN...N2N1-3′-CPG). After capping of the 5′ end, the cyanoethyl
phosphodiester protecting groups are removed by treatment
with NEt3/MeCN at room temperature for 90 min. This is a
necessary step to perform before removal of the 2′-ALE group
to prevent isomerization or cleavage at the branchpoint 3′,5′-
internucleotide linkage. The 2′-ALE groups are then removed
by treatment with buffered 0.5 M hydrazine hydrate at room
temperature for 15 min. The elongation of the last branch from
the 2′ position of nucleotide (A) is then achieved using
commercially available reverse 5′ phosphoramidites, thereby
generating the branched RNA. To force branching at the
sterically hindered 2′-hydroxyl group, both the concentration
and the coupling time of the first 5′-amidite were increased to
0.3 M and 30 min, respectively.30
Synthesis of Native Branched RNAs by Method B. In
contrast to Method A, this method introduces the 2′ branch
segment earlier in the synthesis, immediately after coupling of
the branch-site monomer (B) (Scheme 2B). The first branch is
synthesized in the conventional 3′ to 5′ direction and
completed by the incorporation of synthon (B) at the 5′ end,
5 ‑Levulinyl
producing ′
A
-NN...N2N1-3′-CPG. Next, the cya-
2′‑DMTr
noethyls are cleaved by triethylamine in acetonitrile (2:3 v/v)
and the 2′-DMTr group removed, allowing the growth of a 2′
chain using reverse 5′-phosphoramidites (R). As for method A,
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Table 1. Solid-Phase Synthesis of Branched RNAs
HPLC yield
(%)
isolated yield
(%)
calculated molecular weight calculated exact mass
observed mass
molecule
sequence
UAA(2′GU)CA
UAA(2′GU)CA
method
(Da)
(Da)
(m/z)
I
A
B
Z
A
B
Z
A
B
Z
A
56
54
64
44
46
67
40
45
65
19
25
24
21
26
20
32
11
26
33
6
2188.4
2188.4
2188.4
3491.1
3491.1
3491.1
3491.1
3491.1
3491.1
5090.1
2187.3
2187.3
2187.3
3490.5
3490.5
3490.5
3490.5
3490.5
3490.5
5087.7
2187.3
2187.4
2187.3
3491.5
3491.4
3491.5
3491.7
3491.7
3491.5
5089.2
I
IZ
II
UAaraA(2′GU)CA
UAA(2′GUAUGU)GU
UAA(2′GUAUGU)GU
UAaraA(2′GUAUGU)GU
UAA(2′GU)GUAUGU
UAA(2′GU)GUAUGU
UAaraA(2′GU)GUAUGU
II
IIZ
III
III
IIIZ
IV
UACUAA(2′GUAUG)
CAAGU
IV
UACUAA(2′GUAUG)
B
Z
Z
27
55
62
10
26
18
5090.1
5106.1
5090.1
5087.7
5103.7
5087.7
5089.2
5106.7
5087.7
CAAGU
IVZ
IVZ2
UACUAaraA(2′GUGUG)
CAAGU
UACUAaraA(2′GUAUG)
CAAGU
Figure 2. Anion-exchange HPLC chromatograms of crude synthetic products for branched oligonucleotides I and II, generated by Methods A and B,
and equivalent length syntheses of molecules IZ and IIZ, generated by Method Z.
the concentration and the coupling time of the first 5′-
phosphoramidite is increased to 0.3 M and 30 min, respectively.
Capping and removal of the 5′-levulinyl group from the
branchpoint are carried out in buffered 0.5 M hydrazine hydrate
at room temperature for 15 min, which permits completion of
the Y-shaped branched RNA molecule through the coupling of
standard 3′ phosphoramidite monomers (S). Analogous to our
Method B, branch and hyperbranch RNAs constructed from a
2′-MMTr uridine, 5′-levulinyl, 3′-phosphoramidite branchpoint
synthon37 were effectively prepared by Sabatino and co-workers
for down-regulating GRP78 expression and inducing cell death
in HepG2 liver cancer cells.38
where the 2′ hydroxyl group is cis-oriented with respect to the
nucleobase, and incorporated it at the branching point.
Branched compounds with this modification are not hydrolyzed
by Dbr1 and have been previously shown to inhibit the
debranching enzyme toward native lariat RNAs in pre-mRNA
splicing.39,40 However, until now, the synthetic methodology
employed to generate these compounds required a 2′,3′-bis-O-
phosphoramidite of the arabinose nucleoside that was coupled
simultaneously to two growing RNA chains.39,40 To achieve 2′
and 3′ and regioselectivity at the branchpoint, as well as
increase the overall yield of branched RNA synthesis, our
approach utilizes a novel 2′-O-levulinylated arabinose monomer
(Z) (Scheme 2C). The synthetic strategy for preparing the
arabinose modified bRNA is almost identical to Method A, with
the exception that the decyanoethylation step becomes
Synthesis of Arabinose Modified Branched RNAs. In
addition, we have refined the synthesis of substrates with an
altered configuration at the C2′ of the branchpoint sugar.
Specifically, we have prepared arabinoadenosine derivatives,
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Figure 3. Polyacrylamide gel electrophoresis of 32P-labeled branched oligoribonucleoitdes before and after treatment with Dbr1. (A) Anticipated
debranching reaction of radiolabeled compound II, showing expected products L-II and C-II. (B) Radiolabeled compounds generated by Methods A
and B are indeed substrates of the debranching enzyme. Inhibitor IIZ (generated by Method Z) is not hydrolyzed by the debranching enzyme. The
L-III standard confirms the absence of unintended 2′,3′ isomerization during synthesis, which would not be detectable via mass spectrometry of
isolated bRNA products (Table 1). In addition, this figure can be used to assess the quality of the purified branched products.
unnecessary as the 2′ hydroxyl of the arabinose branchpoint is
no longer cis to the adjacent 3′,5′ phosphotriester linkage.
Optimization of bRNA Synthesis. Four different
branched compounds were prepared using each of the above-
mentioned methods (Table 1). The first was a short heptamer
with two nucleotides branching off from the central adenosine
unit (molecules I and IZ; Table 1). Molecules II and III (as
well as IIZ and IIIZ) were 11-mers, each having 2 nucleotide
units on two branch segments, while the remaining segment
was extended to 6 nucleotides. Finally molecule IV was a 16-
mer with 5 nucleotides on each branch segment.
Preliminary syntheses of these compounds produced the
desired products; however, the HPLC traces of the crude
materials contained significant amounts of failure sequences
(HPLC analysis; Supporting Information Figure S1A). Thus to
augment the yields we sought to synthesize bRNA II via
Method A while modifying several essential synthetic
parameters. Once optimized, these parameters were applied
to Method B as well. Among the parameters tested were
coupling agents (5-ethylthio-1H-tetrazole versus 4,5-dicyanoi-
midazole), deprotection time (delevulination and decyanoe-
thylation), and nucleoside loading on the solid support (CPG).
The products from these trials were assessed by HPLC
(Supporting Information Figure S2). It was found that the
yields were most affected by reduction of the decyanoethylation
time from 3 to 1.5 h and reduction of the delevulination time
from 30 to 15 min. The latter change, which actually increased
yields, might be significant considering the potential risk of loss
of certain base protecting groups upon prolonged treatment
with hydrazine. We were unable to obtain any branched
product with a CPG support having a loading of 78 μmol/g;
however, supports with 30−40 μmol/g loadings afforded
branched products in good yields (Supporting Information
Figure S2). The conditions that were best yielding (Supporting
Information Figure S2) were chosen to resynthesize the bRNAs
listed in Table 1.
(Figure 3; Supporting Information Figures S3 and S4). In terms
of yields, Methods A and B afforded comparable results (Table
1). Longer sequences generally provided less material (6−10%)
than shorter sequences (11−26%), regardless of the method
used (Table 1). Incorporation of arabinose (via Method Z)
afforded slightly cleaner crude products (up to 66% by HPLC)
and higher isolated yields (up to 33%). In Methods A and Z,
and to a lesser extent B, there is one major failure product,
which, on the basis of retention time and mass spectrometry
(Figure 2; see also Supporting Information Figure S5),
corresponds to a linear product that has not undergone
branching at C2′. This unbranched species likely arises from
inefficient coupling of the first 5′-phosphoramidite despite the
higher concentration (0.3 M) used. This results from the
sterically hindered 2′-hydroxyl that is vicinal to a phospho-
diester bond (in the case of branchpoint ribose) or adenine
base (in the case of the arabinose branchpoint), which reduces
overall coupling efficiency.31 This coupling defect was observed
to a lesser extent in Method B, possibly as coupling at the 2′
branch segment takes place before the 5′ segment is
introduced, thereby reducing steric strain at that step.
HPLC analysis of products generated from Methods A and B
reveals the presence of failure products that elute before the
intact bRNA product but after the unbranched side product,
suggesting a significant reduction in the stepwise coupling
yields after initial branchpoint formation. One potential source
of this problem is the decyanoethylation step preceding the
deblocking of a 2′ vicinal hydroxyl. In this step, the cyanoethyl
groups are removed in a triethylamine solution, thus converting
all existing phosphotriesters to phosphodiesters, a mandatory
action that prevents 2′,3′ isomerization and/or strand cleavage
due to nucleophilic attack from the deblocked 2′ vicinal
hydroxyl (Supporting Information Figure S6B).41−43 This has
been observed and solved by decyanoethylation during the
regiospecific solution phase synthesis of branched ribonucleo-
tides.19 However, this process also deblocks cyanoethyl groups
from the phosphotriesters of all previously incorporated
nucleotides. It has been reported that this conversion inhibits
subsequent couplings and reduces the yield of each step from
99 to 93%.44 It was assumed that the main cause of the
coupling inhibition is the presence of several phosphodiester
linkages, which are competing with the 2′ hydroxyl for activated
After synthesis and deprotection, the crude products were
analyzed by anion exchange HPLC (Figure 2, Supporting
Information Figure S1B) and found to contain the expected
full-length products. Isolated in yields ranged from 6 to 26%
(Table 1). The high purity of the isolated bRNAs (77−99%;
average purity: 92%) was confirmed by PAGE and HPLC
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amidite. However, subsequent couplings can be restored to
96−97% efficiency by washing the growing oligonucleotide
with 0.1 M DBU and 0.1 M 1H-tetrazole after each
detritylation step to form a tetrazolium salt with the
phosphate.44 Unfortunately, in our hands, this protocol failed
to improve postbranching coupling yields (Supporting
Information Figure S2). Evidently, this limitation does not
affect synthesis of the arabinose bRNAs as it lacks the
decyanoethylation step, since the arabinose 2′ hydroxyl is
trans to the 3′ phosphotriester and prevents 2′-3′ isomerization
or strand cleavage.
Characterization of Branched Compounds by Ex-
posure to Debranching Enzyme (Dbr1). Samples of
branched RNA II (synthesized by Methods A and B) were
exposed to a recombinant preparation of the intron
debranching enzyme from Entamoeba histolytica (Dbr1) in
order to determine the efficacy of this compound as a mimic of
cellular lariat and branched RNAs. The samples were
radiolabeled at the 5′ position in order to assay the debranching
reaction in vitro using denaturing polyacrylamide gel electro-
phoresis. In this assay, only one of the two products is visible
after debranching and has a significantly enhanced migration
through the gel relative to the unhydrolyzed substrate (Figure 3
and Supporting Information Figure S3). The resulting data
confirm bRNA II is bona fide substrate of the enzyme. As
expected from previous studies, the arabinoadenosine branched
derivative IIZ is not hydrolyzed by the enzyme. These findings
are consistent with other preparations of bRNA made
previously in our lab and elsewhere.39,40
In addition, some of the products of hydrolysis were purified
by HPLC (Figure 4 and Supporting Information Figure S7)
and their composition analyzed by MS (Supporting Informa-
tion Figure S7). The data confirm these products match the
anticipated masses for RNA chains independent of the
synthetic methods used, confirming Methods A and B are
regioselective around the branchpoint.
Inhibition of Dbr1 by Arabinose Branched Analogues.
As the product of Method Z is not hydrolyzed by Dbr1, and
similar compounds have also been shown to inhibit the
enzyme,39,40 we evaluated the effect of increasing concen-
trations of molecules IZ and IVZ on debranching activity
toward a native branchpoint. In this approach, molecule II
(generated by Method A) was 32P-labeled and exposed to
Dbr1, and the debranching products were monitored by
denaturing PAGE electrophoresis. Concentrations of IZ or IVZ
and linear control UAACA (L-I) were increased from 0 to 32
μM (Figure 5 and Supporting Information Figure S8). The
results were subsequently subjected to densitometry calcu-
lations, from which the maximal reaction progression was
determined. The resulting data show that both arabinose
molecules IZ and IVZ inhibit Dbr1 and that IVZ is apparently
more potent.
Figure 4. Anion-exchange chromatograms of substrate I before and
after treatment with debranching enzyme Dbr1. Near-complete
debranching is observed for substrates made by Methods A and B.
It was not feasible to measure an experimental mass for purified
product C-I.
products of the correct length and revealed that the
arabinoadenosine compound made by this approach can inhibit
debranching activity. These findings will aid in the future
development and large-scale production of therapeutic bRNA
compounds.
EXPERIMENTAL SECTION
■
Solid Phase Synthesis of Branched RNAs. Materials and
Reagents. Branched oligonucleotide syntheses were carried out using
a DNA/RNA synthesizer and a universal (UnyLink) solid support on a
1 μmol scale (loading 29.9 μmol/g). Conventional and reverse 2′
TBDMS and bis-cyanoethyl-N,N-diisopropyl phosphoramidite were
used (0.15 M in MeCN). Phosphoramidites were dissolved in MeCN
and activated with 5-ethylthio-1H-tetrazole (0.25 M in MeCN).
Capping was carried out by the simultaneous delivery of acetic
anhydride in pyridine/THF and N-methylimidazole (16% in THF)
and contacting the solid support for 6 s. Oxidation of phosphite
triester intermediates was effected with 0.1 M iodine in pyridine/H2O/
THF (20 s). A solution of 3% trichloroacetic acid in THF, delivered
over 1.8 min, was used to deblock DMTr groups. Anhydrous
triethylamine/MeCN (2:3 v/v) and 0.5 M hydrazine hydrate in
pyridine/acetic acid (3:2 v/v) were used to remove, respectively,
cyanoethyl (CNEt) phosphate and Levulinyl/ALE protecting groups.
Oligonucleotides were deblocked and released from the solid support
using a concentrated ammonium hydroxide solution. TBDMS groups
were cleaved with TREAT-HF.
CONCLUSIONS
■
In summary, we have synthesized various bRNAs of distinct
sequences using two different approaches. An additional
approach was developed to generate branched compounds
containing an arabinose branchpoint. The purity of the crude
products varied between the methods and the compounds:
Method Z, with an arabinose branchpoint, generated the
highest yield of branched compound, likely because it avoids a
decyanoethylation step that reduces the yields of all subsequent
coupling steps. Exposure to Dbr1 resulted in hydrolysis
Method A (Scheme 2A). 5′-NN′...N2′N1′-A2′‑ALE-NN...N2N1-3′ was
grown on the solid support (1 μmol columns) in the conventional 3′
to 5′ direction using standard 3′ RNA phosphoramidites (denoted as S
in Scheme 2). The A2′‑ALE unit in this sequence was introduced by
coupling monomer (A) for 10 min (0.13 M in MeCN, activator: 5-
ethylthio-1H-tetrazole). Subsequently the 5′-DMTr was removed, and
the deblocked hydroxyl was capped affording a 5′-O-acetylated linear
oligomer. Anhydrous triethylamine/MeCN (2:3 v/v) solution was
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Figure 5. bRNAs with arabinoadenosine branchpoints are effective inhibitors of Dbr1. (A) Anticipated debranching reaction for substrate II, which
contains a natural adenosine branchpoint. (B) The arabinoadenosine compounds inhibit debranching of substrate II in a dose-dependent fashion.
The larger arabinoadenosine compound IVZ is apparently a more effective inhibitor than the smaller compound IZ. A similar titration series with
product analogue 5′-UAACA-3′ (L-I) was used to ensure the observed inhibition is specific to the arabinoadenosine branchpoint (Supporting
Information Figure S8).
passed through the solid support and allowed to react for 20 min. This
step was repeated four times to ensure complete removal of the
cyanoethyl phosphodiester protecting groups. After washing with
MeCN (5 min) and drying the support over Ar stream (10 min), the
synthesis columns were temporarily removed from the synthesizer and
placed in a round-bottom flask under a high vacuum for one hour. The
columns were placed back on the synthesizer, and a freshly prepared
solution of 0.5 M hydrazine hydrate in pyridine/acetic acid (3:2 v/v)
was flowed through the columns for 20 s and allowed to react for 3.75
additional minutes. This step was repeated four times to ensure
complete removal of the 2′-ALE groups. After washing (MeCN, 10
min) and drying (Ar gas, 10 min), the solid supports were dried under
a high vacuum (1 h). The synthesis columns containing the solid
supports were returned to the synthesizer, and a reverse phosphor-
amidite corresponding to N1″ position (0.3 M in MeCN) was coupled
for 30 min. The remaining units were coupled for 15 min at the same
concentration, and completion of this branch segment afforded the
min) and drying (Ar gas, 10 min), the solid supports were dried under
a high vacuum (1 h). The synthesis columns containing the solid
supports were returned to the synthesizer, and 3′-5′ synthesis of units
N1′N2′...NN′ resumed. For the final branch segment the phosphor-
amidites were dissolved in 0.3 M MeCN, and coupling time was set to
30 min for the first unit and 15 min for the remaining ones.
Method Z (Scheme 2C). 5′-NN′...N2′N1′‑AraA2′‑Levulinyl−NN...N2N1-3′
was grown on the solid support (1 μmol columns) in the conventional
3′ to 5′ direction using standard 3′ RNA phosphoramidites (denoted
as S in Scheme 2). The A2′‑Levulinyl unit in this sequence was
Ara
introduced by coupling monomer (Z) for 10 min (0.13 M in MeCN,
activator: 5-ethylthio-1H-tetrazole). Subsequently the 5′-DMTr was
removed, and the deblocked hydroxyl was capped affording a 5′-O-
acetylated linear oligomer. The sample was then washed with MeCN
(5 min), and the support was dried over Ar stream (10 min).
Subsequently, a freshly prepared solution of 0.5 M hydrazine hydrate
in pyridine/acetic acid (3:2 v/v) was flowed through the columns for
20 s and allowed to react for 3.75 additional minutes. This step was
repeated four times to ensure complete removal of the 2′-Levulinyl
groups. After washing (MeCN, 10 min) and drying (Ar gas, 10 min),
the solid supports were dried under a high vacuum (1 h). The
synthesis columns containing the solid supports were returned to the
synthesizer, and a reverse phosphoramidite corresponding to N1″
position (0.3 M in MeCN) was coupled for 30 min. The remaining
units were coupled at 0.15 M for 15 min, and completion of this
branch segment afforded the fully protected branched RNA analogues.
Deprotection. Solid supports were transferred into a screw cap
centrifuge tube into which 250 μL of anhydrous ethanol and 750 μL
cold (−20 °C) concentrated ammonia were added. The samples were
shaken for 48 h at room temperature to simultaneously remove the
remaining cyanoethyl groups, deblock base protecting groups, and
release the oligonucleotides from the support. Samples were
centrifuged for 10 min at 11000g. The supernatant was cooled over
dry ice for 5 min and evaporated using a speedvac evaporation system.
TREAT-HF (150 μL/μmol of branched RNA) was added, and the
solution was shaken for 48 h at room temperature. The crude
branched compounds were isolated by adding cold n-butanol (1 mL,
−20 °C), followed by 3 M NaOAc (30 μL; pH 5.5), and centrifuging
the resulting mixture at 11000g. After removing the supernatant, the
fully protected branched RNAs.
5 ‑Levulinyl
Method B (Scheme 2B). ′
A
-NN...N2N1-3′ was grown
2′‑DMTr
on the solid support (1 μmol columns) in the conventional 3′ to 5′
5 ‑Levuliny-
direction using standard 3′ phosphoramidites (S). The
′
lA2′‑DMTr unit in this sequence was introduced by coupling monomer
(B) for 15 min (0.15 M in MeCN, activator: 5-ethylthio-1H-tetrazole).
Anhydrous triethylamine/MeCN (2:3 v/v) solution was passed
through the solid support and allowed to react for 20 min. This
step was repeated four times to ensure complete removal of the
cyanoethyl phosphotriester protecting groups. After washing with
MeCN (5 min) and drying the support over Ar stream (10 min), the
synthesis columns were temporarily removed from the synthesizer and
placed in a round-bottom flask under a high vacuum for one hour. The
columns were placed back on the synthesizer, and the growing
oligonucleotide was then detritylated and extended by units
N1″N2″...NN″ in the 5′ to 3′ direction. Subsequently the 3′-DMTr
was removed, and the deblocked hydroxyl was capped affording a 3′-
O-acetylated V-shaped oligomer. Subsequently, a freshly prepared
solution of 0.5 M hydrazine hydrate in pyridine/acetic acid (3:2 v/v)
was flowed through the columns for 20 s and allowed to react for 3.75
additional minutes. This step was repeated four times to ensure
complete removal of the 2′-ALE groups. After washing (MeCN, 10
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Article
remaining pellet (which contained the bRNA) was dried, taken up in
DEPC-treated autoclaved water (1 mL), and filtered.
were heated to 95 °C and loaded onto a 16% polyacrylamide gel with
7 M Urea and Tris-Borate-EDTA buffer in an electrophoresis
apparatus. After 2−3 h at 1000 V, the gel was frozen in a cassette
with an X-ray film overnight, and developed.
Inhibition Assays and Analysis by Autoradiography. Inhibition
reactions were performed at 28 nM substrate, 10 nM enzyme and an
identical buffer to the above debranching assays. Reactions were
carried out in the presence of increasing concentrations of inhibitors
(0, 2, 4, 8, 16, and 32 μM) and stopped 2 min into the reaction by
addition of an equal volume of formamide loading buffer. The samples
were loaded on the gel in ascending order of inhibitor concentration
(Figure 5 and Supporting Information Figure S8).
Autoradiography was performed as above. The films were scanned,
and densitometry was performed using an image processing program
(ImageJ). The inhibition data were fit by nonlinear regression to a
simple exponential decay function of form y = e−kx + C using the
program GraphPad Prism.
Purification. Branched compounds were purified by HPLC
(chromatograms not shown) using an anion exchange column
(Protein PAK DEAE 5PW 21.5 mm × 15 cm). The buffer system
consisted of water (solution A) and 1 M aq lithium perchlorate
(solution B), at a flow rate of 4 mL/min. The gradient was 0−40% B
over 50 min at 60 °C. Under these conditions the desired peaks eluted
at 25−35 min. The collected samples were lyophilized and redissolved
in 1 mL of autoclaved Milli-Q H2O and loaded into a pre-eluted size
exclusion column filled with NAP-25 matrix. The column was eluted
with more Milli-Q H2O and 7 × 1 mL fractions were collected. Each of
these fractions was quantified by absorbance (λ = 260 nm) and assayed
for salt content (λ < 230 nm). To obtain the number of nanomoles, a
conversion factor was used, (12.99 nmol/OD for bRNA I, 8.42 nmol/
OD for bRNAs II, IIZ, III, IIIZ and 6.04 for bRNA IV and IVZ) as
calculated for a linear RNA of the same sequence by the IDT DNA
oligoanalyzer program.45 The masses of the molecules were confirmed
by LC−MS ESI-Q-TOF or ESI-TOF (Table 1, Supporting
Information Figure S9). The quality of the purified products was
also assessed by reinjection into the HPLC (Supporting Information
Figure S4).
Oligonucleotide Handling. Before and after purification, the
oligonucleotides were handled with sterilized (16 psi, 125 °C 30
min) pipet tips, microcentrifuge tubes, Sephadex (size-exclusion NAP-
25 columns), and diethylpyrocarbonate (DEPC) treated water (less
than 1 month old). Oligonucleotides were then typically stored at −20
°C.
Radiolabeling the bRNA Samples. 100 pmol of sample was reacted
with γ-32P-ATP in presence of T4 polynucleotide kinase (2000 units)
and 1× PNK buffer (70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT,
pH 7.6 at 25 °C). The reaction was carried out in a total volume of 20
μL at 37 °C for 1 h. After completion of the reaction, the mixture was
loaded into a size-exclusion column (NAP-10), eluted with sterilized
water, and collected as 500 μL fractions. The early most radioactive
fractions were combined, evaporated, and redissolved to 20 000 CPM/
μL.
Analytical HPLC. The crude samples as well as the products of the
debranching assays were assayed on HPLC using an anion exchange
column (Protein DEAE 5PW 7.5 × 75 mm). The buffer system
consisted of water (solution A) and 1 M aq lithium perchlorate
(solution B), at a flow rate of 1 mL/min. The gradient was 0−24%
solution B over 30 min at 65 °C Under these conditions the desired
peaks eluted at roughly 18 min for compound I and 21.5 min for
compounds II, IIZ, III, and IIIZ (Figure 2 and Supporting
Information Figures S1, S2, and S5).
Expression and Purification of Debranching Enzyme. A codon-
optimized version of the DBR1 gene from Entamoeba histolytica was
cloned into a modified pET15b plasmid for expression of a
hexahistidine-tagged protein in Escherichia coli. The protein was
purified using immobilized metal ion chromatography and cation
exchange chromatography. SDS-PAGE was used to confirm the purity
of the protein used in activity assays. A more detailed purification
protocol is to be reported elsewhere (Montemayor et al., manuscript
in preparation). The purified protein was found to be very sensitive to
repeated cycles of freezing and thawing, with an approximate 2-fold
decrease in observed debranching activity for each freeze/thaw cycle.
Debranching Assays and Analysis by HPLC. Purified branched
RNA (10 nmol) was digested by 20 pmol of debranching enzyme in
100 μL debranching buffer (50 mM NaCl, 10 mM HEPES, 10 mM
MnCl2, 10% glycerol, 1 mM tris(2-carboxyethyl)phosphine (TCEP),
pH 7.0) for 2 h. The reaction mixture was directly subjected to anion
exchange HPLC purification and peaks collected, dried down, and
subsequently analyzed by mass spectrometry.
Synthesis of the Branchpoint Monomer (A) for Method A
(Scheme 1A). The synthesis of N6-benzoyl-2′-O-(acetyl levulinic
ester)-3′-O-[(2-cyanoethyl-N,N-diisopropyl) phosphoramidite]-5′-O-
(4,4′-dimethoxytrityl)-adenosine (A) was performed according to a
previous published method35 with slight modifications.
N6-Benzoyl-2′-O-[(methylthio)methyl]-3′,5′-O-[1,1,3,3-tetrakis(1-
methylethyl)-1,3-disiloxanediyl]-adenosine (1). Title compound was
synthesized according to previously published protocols.34
N6-Benzoyl-2′-O-[acetal levulinic ester]-3′,5′-O-[1,1,3,3-tetrakis-
(1-methylethyl)-1,3-disiloxanediyl]-adenosine (2). CsHCO3 (582
mg, 3 mmol) was suspended in DMF (2.5 mL), levulinic acid
(0.625 mL, 6.1 mmol) was added, and the mixture was heated to 75
°C for 3 h. Compound 1 (1.33 g, 1.97 mmol) was dissolved in CH2Cl2
(20 mL) and cooled to 0 °C under Ar. Freshly made SO2Cl2 (10 mL,
1 M in CH2Cl2, 10 mmol) was added dropwise over 20 min, and the
mixture was stirred at 0 °C for 1 h and then 2 h at room temperature.
The mixture was concentrated under N2, redissolved in CH2Cl2 (15
mL), and cannulated to the Cs-activated levulinic acid. The flask was
washed with CH2Cl2 (5 mL), and the reaction mixture was stirred at
room temperature. After 15 h, CH2Cl2 and NaHCO3 (sat aq) were
added, and the mixture was separated. The organic phase was washed
twice with NaHCO3 and subsequently once with brine. The combined
aqueous layers were extracted 3 times with CH2Cl2; the combined
organic extracts were washed once with brine and subsequently
combined with the original organic layer. The combined organic layers
were dried with MgSO4, filtered, and concentrated. The residue was
purified by column chromatography (SiO2, 1:1→1:2 Hexanes/EtOAc)
1
to give compound 2 (871 mg, 59%) as an amorphous white solid: H
NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.63 (d, J 2.3 Hz, 1H),
8.52 (s, 1H), 8.05−7.99 (m, 2H), 7.63 (t, J 7.4 Hz, 1H), 7.53 (t, J 7.6
Hz, 2H), 6.10 (s, 1H), 5.47 (d, J 6.5 Hz, 1H), 5.37 (d, J 6.5 Hz, 1H),
5.07 (dd, J 9.0 Hz, 5.2, 1H), 4.93 (d, J 5.3 Hz, 1H), 4.07−4.01 (m,
1H), 3.98−3.89 (m, 2H), 2.63 (t, J 6.4 Hz, 2H), 2.47−2.35 (m, 2H),
1.97 (s, 2H), 1.12−0.96 (m, 28H); 13C NMR (75 MHz, DMSO-d6) δ
209.9, 206.9, 175.7, 171.9, 166.1, 152.5, 152.1, 151.0, 143.5, 133.7,
132.9, 128.95, 128.91, 126.2, 98.9, 88.6, 86.9, 86.1, 81.3, 71.1, 61.3,
37.4, 29.8, 27.6, 17.74, 17.72, 17.69, 17.66, 17.54, 17.49, 13.58, 13.57,
13.4, 13.3, 13.21, 13.16, 12.8; HRMS (ESI Q-TOF) calcd for
C35H52N5O9Si2 (M + H)+ 742.3304, found 742.3298.
N6-Benzoyl-2′-O-[acetal levulinic ester]-adenosine (3). Com-
pound 2 (2.61 g, 3.46 mmol) was dissolved in THF (8.7 mL) and
stirred at room temperature under Ar. TREAT-HF (1.0 mL, 1.84
mmol) was added. After 2.5 h the mixture was diluted with 100 mL of
CH2Cl2 and concentrated. The residue was purified by column
chromatography (SiO2, 1:200 → 1:25 CH3OH/CH2Cl2) to yield
1
compound 3 (1.20 g, 73%) as an amorphous white solid: H NMR
(300 MHz, acetone-d6) δ 10.20 (s, 1H), 8.63−8.69 (m, 1H), 8.56−
8.63 (m, 1H), 8.12 (dd, J 9.8, 8.6 Hz, 2H), 7.48−7.69 (m, 3H), 6.21 (t,
J 6.4 Hz, 1H), 5.18−5.38 (m, 3H), 5.01 (dd, J 6.2, 4.9 Hz, 1H), 4.45−
4.69 (m, 2H), 4.19 (q, J 2.5 Hz, 1H), 3.66−3.96 (m, 2H), 3.06 (s, 1H),
2.55−2.69 (m, 2H), 2.21−2.34 (m, 2H), 2.01−2.10 (m, 3H); 13C
NMR (101 MHz, CDCl3) δ 207.5, 183.1, 172.0, 164.9, 152.1, 150.6,
150.3, 143.5, 133.3, 133.0, 128.8, 128.0, 124.3, 89.0, 88.9, 87.9, 82.6,
Debranching Assays and Analysis by Autoradiography. Reac-
tions were carried out at 2 μM substrate concentrations (including 800
CPM/μL radioactive substrate), 10 nM enzyme in debranching buffer
(100 mM NaCl, 20 mM HEPES, 10% glycerol, 1 mM TCEP, 0.1 mM
MnCl2, pH 7.0). Aliquots were removed and added to an equal volume
of formamide gel loading buffer to quench the reaction. The products
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71.8, 63.0, 37.8, 29.7, 27.7; HRMS (ESI Q-TOF) calcd for
C23H25N5O8Na (M + Na)+ 522.1601, found 522.1585.
Dimethoxytrityl chloride (4.73 g, 13.9 mmol) was added, and the
reaction was stirred at room temperature. After 1.2 h, CH2Cl2 and
NaHCO3 (sat aq) were added, and the mixture was separated. The
organic phase was washed twice with NaHCO3 and once with brine.
The combined aqueous layers were extracted 3 times with CH2Cl2,
and the combined organic extracts were washed once with brine. They
were subsequently combined with the original organic layer, dried with
MgSO4, filtered, and concentrated. The crude syrup was coevaporated
with toluene to yield N6-benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′,5′-O-
[1,1,3,3-tetrakis(1-methylethyl)-1,3-disiloxanediyl]-adenosine.
N6-Benzoyl-2′-O-[acetal levulinic ester]-5′-O-[4,4′-dimethoxytri-
tyl]-adenosine (4). Compound 3 (508 mg, 1.02 mmol) and AgNO3
(206 mg, 1.21 mmol) were dissolved in THF (4.95 mL) and pyridine
(0.55 mL) and stirred under Ar. 4,4′-Dimethoxytrityl chloride (411
mg, 1.21 mmol) was added, and the reaction was stirred at room
temperature. After 1.2 h, CH2Cl2 and NaHCO3 (sat aq) were added,
and the mixture was separated. The organic phase was washed twice
with NaHCO3 and once with brine. The combined aqueous layers
were extracted 3 times with CH2Cl2, and the combined organic
extracts were washed once with brine. They were subsequently
combined with the original organic layer, dried with MgSO4, filtered,
and concentrated. The residue was purified by column chromatog-
raphy (SiO2, 1:200 → 3:50 CH3OH/CH2Cl2) to yield compound 4
The crude N6-benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′,5′-O-[1,1,3,3-
tetrakis(1-methylethyl)-1,3-disiloxanediyl]-adenosine was dissolved in
THF (29 mL), and TREAT-HF (3.3 mL, 20 mmol) was added. After
2.5 h, CH2Cl2 and NaHCO3 (sat aq) were added, and the mixture was
separated. The organic phase was washed twice with NaHCO3 and
once with brine. The combined aqueous layers were extracted 3 times
with CH2Cl2, and the combined organic extracts were washed once
with brine and subsequently combined with the original organic layer.
The combined organic phase was dried with MgSO4, filtered, and
concentrated. The residue was purified by column chromatography
(SiO2, 1:200 → 3:50 CH3OH/CH2Cl2) to yield compound 7 (4.34 g,
55% over 2 steps) as an amorphous white solid: 1H NMR (300 MHz,
acetone-d6) δ 10.45 (s, 1H), 8.48 (s, 1H), 8.46 (s, 1H), 8.16 (d, J 7.3
Hz, 2H), 7.47−7.64 (m, 3H), 7.41 (dd, J 8.9, 7.5 Hz, 2H), 7.28 (d, J
8.9 Hz, 2H), 7.07−7.20 (m, 5H), 6.73 (d, J 8.9 Hz, 2H), 6.61 (d, J 8.9
Hz, 2H), 6.33 (d, J 7.6 Hz, 1H), 5.55 (d, J 8.6 Hz, 1H), 5.12 (dd, J 7.6
Hz, 4.3, 1H), 4.08 (s, 1H), 3.81 (d, J 1.9 Hz, 1H), 3.69 (d, J 13.9 Hz,
7H), 3.45 (t, J 10.5 Hz, 1H), 3.32 (s, 1H), 3.21 (s, 1H); 13C NMR (75
MHz, acetone-d6) δ 165.4, 158.9, 158.6, 151.3, 151.1, 150.8, 145.3,
143.9, 135.5, 135.3, 134.1, 132.5, 130.3, 130.1, 129.6, 128.6, 128.4,
127.8, 127.7, 126.8, 125.8, 113.0, 112.9, 88.8, 87.8, 87.3, 75.7, 71.6,
62.8, 54.7, 54.7; HRMS (ESI Q-TOF) calcd for C38H36N5O7 (M +
H)+ 674.2615, found 674.2599.
1
(653 mg, 79%) as an amorphous white solid: H NMR (300 MHz,
acetone-d6) δ 8.61 (s, 1H), 8.49 (d, J 6.4 Hz, 1H), 8.12 (d, J 7.3 Hz,
2H), 7.43−7.71 (m, 5H), 7.13−7.41 (m, 7H), 6.84 (dd, J 8.9 Hz, 3.1,
4H), 6.29 (d, J 4.7 Hz, 1H), 5.40 (dd, J 26.9, 6.5 Hz, 2H), 5.21 (t, J 4.9
Hz, 1H), 4.79 (d, J 4.2 Hz, 1H), 4.27 (q, J 4.1 Hz, 1H), 3.75 (s, 6H),
3.47 (dd, J 13.1, 6.4 Hz, 2H), 2.66 (t, J 6.4 Hz, 2H), 2.37 (t, J 6.4 Hz,
2H), 2.07 (d, J 9.0 Hz, 3H); 13C NMR (75 MHz, acetone-d6) δ 206.0,
171.9, 158.7, 152.1, 151.8, 150.4, 145.1, 143.0, 135.9, 135.8, 134.2,
132.3, 130.1, 128.5, 128.3, 128.1, 127.7, 126.7, 125.1, 113.0, 88.4, 87.2,
86.2, 84.0, 81.2, 70.2, 63.5, 54.6, 37.2, 28.4, 27.6; HRMS (ESI Q-TOF)
calcd for C44H43N5O10Na (M + Na)+ 824.2908, found 824.2911.
N6-Benzoyl-2′-O-(acetyl levulinic ester)-3′-O-[(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite]-5′-O-(4,4′-dimethoxytrityl)-adeno-
sine (A). Compound 4 (567 mg. 0.71 mmol) was dissolved in 4.24 mL
THF, and the mixture was stirred at room temperature under Ar. N,N-
Diisopropylethylamine (0.9 mL, 5.58 mmol) was added, and
subsequently 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(0.195 mL, 0.78 mmol) was added. After 4 h, CH2Cl2 and NaHCO3
(sat aq) were added, and the mixture was separated. The organic phase
was washed twice with NaHCO3 and once with brine. The combined
aqueous layers were extracted 3 times with CH2Cl2, and the combined
extracts were washed once with brine. Subsequently the extracts were
combined with the original organic layer, dried with MgSO4, filtered,
and concentrated. The residue was purified by column chromatog-
raphy (SiO2, 1:2 hexanes/EtOAc + 1% NEt3) to yield compound 5
N6-Benzoyl-2′-O-(4,4′-dimethoxytrityl)-5′-O-levulinyl-adenosine
(8). O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethylammonium tetra-
fluoroborate (TBTU, 3.02 g, 9.45 mmol) was dissolved in DMF
(22.7 mL) and N,N-diisopropylethylamine (3.3 mL) and stirred at
room temperature under Ar. Freshly distilled levulinic acid (0.96 mL,
9.45 mmol) was added, and the reaction was stirred at room
temperature for 30 min. Compound 7 (2.12 g, 3.15 mmol) was dried
under a vacuum in a round-bottom flask with a magnetic stirrer. Ten
milliliters of the TBTU/levulinic acid reaction was added to the
nucleoside and stirred at room temperature for 2 h. Subsequently the
remainder of the TBTU/levulinic acid reaction mixture was
cannulated to the nucleoside, and the reaction was stirred at room
temperature. After 10 h, CH2Cl2 and NaHCO3 (sat aq) were added,
and the mixture was separated. The organic phase was washed twice
with NaHCO3 and once with brine. The combined aqueous layers
were extracted 3 times with CH2Cl2. Next, the combined organic
extracts were washed once with brine and subsequently combined with
the original organic layer, dried with MgSO4, filtered, and
concentrated. The residue was purified by column chromatography
(SiO2, 1:200 → 1:50 CH3OH/CH2Cl2) to yield compound 8 (1.44 g,
84%) as an amorphous white solid. There was a difficulty fully
purifying the compound from the starting material; therefore, the
NMR analysis is that of the purest fraction, but the entire material was
1
(508 mg, 71%) as an amorphous white solid: H NMR (300 MHz,
acetone-d6) δ 8.62 (d, J 2.5, 1H), 8.53 (d, J 1.2 Hz, 1H), 8.07−8.20
(m, 2H), 7.04−7.75 (m, 12H), 6.80−6.92 (m, 4H), 6.31 (t, J 5.0 Hz,
1H), 5.29−5.53 (m, 2H), 4.96−5.09 (m, 1H), 3.79−4.46 (m, 1H),
3.76 (d, J 2.2 Hz, 7H), 3.34−3.74 (m, 6H), 2.59−2.96 (m, 4H), 2.38
(dt, J 9.9, 6.4 Hz, 2H), 1.18−1.34 (m, 12H), 1.13 (t, J 8.2 Hz, 3H);
13C NMR (75 MHz, acetone-d6) δ 206.0, 205.9, 172.0, 171.8, 158.7,
152.1, 152.0, 151.8, 150.5, 145.10, 145.06, 143.4, 143.2, 135.83,
135.81, 135.75, 134.2, 132.4, 130.2, 130.10, 130.07, 128.5, 128.32,
128.26, 128.2, 128.1, 127.73, 127.71, 126.72, 126.69 125.3, 125.2,
118.3, 118.0, 113.0, 88.5, 88.3, 87.9, 87.4, 86.29, 86.26 83.6, 83.2, 83.1,
80.3, 79.8, 79.7, 72.5, 71.7, 71.5, 71.4, 71.1, 63.1, 62.9, 61.1, 59.4, 59.1,
58.7, 58.5, 58.3, 58.2, 54.68, 54.67 45.0, 44.9, 43.22, 43.16 43.1, 43.0,
37.17, 37.15 27.7, 27.6, 24.24, 24.21, 24.1, 24.0, 22.32, 22.29, 22.26,
22.2, 20.0, 19.9, 19.8, 19.5, 19.4; 31P NMR (81 MHz, DMSO-d6) δ
149.5, 149.0, 13.9, −2.0; HRMS (ESI Q-TOF) calcd for
C53H61N7O11P (M + H)+ 1002.4167, found 1002.4160.
1
carried through to the next step: H NMR (400 MHz, DMSO-d6) δ
11.20 (s, 1H), 8.56 (s, 1H), 8.35 (s, 1H), 8.10−7.99 (m, 2H), 7.63 (d,
J 7.3 Hz, 1H), 7.54 (t, J 7.5 Hz, 2H), 7.38 (dd, J 6.0, 2.3 Hz, 2H), 7.25
(d, J 8.9 Hz, 2H), 7.13 (ddd, J 14.7, 11.5, 4.2 Hz, 5H), 6.70 (d, J 9.0
Hz, 2H), 6.60 (d, J 9.0 Hz, 2H), 5.86 (d, J 5.4 Hz, 1H), 5.40 (d, J 5.9
Hz, 1H), 4.98 (t, J 5.3 Hz, 1H), 4.07−4.22 (m, 2H), 3.92−4.04 (m,
1H), 3.64 (d, J 14.5 Hz, 6H), 3.43 (d, J 4.6 Hz, 1H), 2.62 (t, J 6.5 Hz,
2H), 2.28−2.41 (m, 2H), 2.04 (s, 3H); 13C NMR (75 MHz, acetone-
d6) δ 205.8, 205.5, 171.9, 165.3, 159.0, 158.8, 151.6, 150.4, 145.3,
143.3, 136.1, 135.6, 135.3, 134.1, 132.4, 130.4, 130.3, 130.0, 129.8,
129.6, 129.2, 128.5, 128.3, 128.1, 127.8, 127.7, 127.6, 126.9, 126.5,
125.4, 113.1, 113.0, 112.9, 112.7, 87.3, 82.7, 75.4, 70.7, 63.7, 54.7, 37.9,
37.3, 28.8, 27.5; HRMS (ESI Q-TOF) calcd for C43H41N5O9Na (M +
Na)+ 794.2802, found 794.2782.
Synthesis of the Branchpoint (B) for Method B (Scheme 1B).
The synthesis of N6-benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′-O-[(2-
cyanoethyl-N,N-diisopropyl) phosphoramidite]-5′-O-levulinyl-adeno-
sine (B) was done following a published synthesis of the analogous
guanosine36 (N6-benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′-O-[(2-cya-
noethyl-N,N-diisopropyl) phosphoramidite]-5′-O-levulinyl-guano-
sine).
N6-Benzoyl-3′,5′-O-[1,1,3,3-tetrakis(1-methylethyl)-1,3-disiloxa-
nediyl]-adenosine (6). Title compound was made according to
previously established protocols.46
N6-Benzoyl-2′-O-(4,4′-dimethoxytrityl)-adenosine (7). Compound
6 (7.14 g, 11.6 mmol) and AgNO3 (2.37 g, 13.9 mmol) were dissolved
in THF (52 mL) and pyridine (5.8 mL) and stirred under Ar. 4,4′-
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N6-Benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′-O-[(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite]-5′-O-levulinyl-adenosine (B). Com-
pound 8 (1.44 g. 1.86 mmol) was dissolved in 11.1 mL THF, and
the mixture was stirred at room temperature under Ar. N,N-
Diisopropylethylamine (2.35 mL, 14.57 mmol) was added, followed
by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.46 mL, 2.1
mmol). After 16 h, CH2Cl2 and NaHCO3 (sat aq) were added, and the
mixture was separated. The organic phase was washed twice with
NaHCO3 and once with brine. The combined aqueous layers were
extracted 3 times with CH2Cl2. Next the combined organic extracts
were washed once with brine and subsequently combined with the
original organic layer, dried with MgSO4, filtered, and concentrated.
The residue was purified by column chromatography (SiO2, CH2Cl2 +
1% NEt3) to yield compound 9 (1.00 g, 56%) as an amorphous white
stirred at room temperature. After 2.5 h the mixture was concentrated.
The residue was purified by column chromatography (SiO2, 1:100 →
1:20 CH3OH/CH2Cl2) to yield compound 12 (2.37 g, 75%) as an
1
amorphous white solid: H NMR (400 MHz, DMSO-d6) δ 11.20 (s,
1H), 8.73 (s, 1H), 8.57−8.64 (m, 1H), 7.98−8.08 (m, 2H), 7.44−7.69
(m, 3H), 6.58 (d, J 5.8 Hz, 1H), 5.88 (s, 1H), 5.35 (t, J 5.9 Hz, 1H),
5.11 (s, 1H), 4.46 (s, 1H), 3.90 (ddd, J 6.6, 4.9, 3.5 Hz, 1H), 3.71
(ddd, J 17.2, 12.2, 4.2 Hz, 2H), 2.36 (t, J 6.7 Hz, 2H), 2.18−2.29 (m,
1H), 1.98 (dd, J 11.7, 5.2 Hz, 1H), 1.94 (d, J 3.0 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 206.5, 171.5, 166.0, 152.4, 152.2, 150.7, 143.7,
133.8, 132.9, 128.9, 125.8, 83.6, 81.8, 77.9, 72.0, 60.7, 37.4, 29.7, 27.6;
HRMS (ESI Q-TOF) calcd for C22H23N5O7Na (M + Na)+ 492.1495,
found 492.1487.
N6-Benzoyl-2′-O-levulinyl-5′-O-[4,4′-dimethoxytrityl]-adenosine-
9-β-D-arabinofuranoside (13). Compound 12 (2.24 g, 4.76 mmol)
was dissolved in pyridine (34 mL) and stirred under Ar. 4,4′-
Dimethoxytrityl chloride (1.78 g, 5.24 mmol) was added, and the
reaction was stirred at room temperature. After 10 h, NaHCO3 (sat aq,
30 mL) was added, and the reaction mixture placed on a rotary
evaporator to remove most of the solvent. Subsequently, CH2Cl2 and
NaHCO3 (sat aq) were added, and the mixture was separated. The
organic phase was washed twice with NaHCO3 and once with brine.
The combined aqueous layers were extracted 3 times with CH2Cl2,
and the combined organic extracts were washed once with brine.
Subsequently, the extracts were combined with the original organic
layer, dried with MgSO4, filtered, and concentrated. The residue was
purified by column chromatography (SiO2, 1:100 → 1:20 CH3OH/
CH2Cl2) to yield compound 13 (3.30 g, 90%) as an amorphous white
1
solid: H NMR (300 MHz, DMSO-d6) δ 8.55 (dd, J 21.4, 19.9 Hz,
1H), 8.05 (d, J 7.2 Hz, 2H), 7.61−7.68 (m, 1H), 7.52−7.59 (m, 2H),
6.96−7.30 (m, 10H), 6.73 (dd, J 13.0, 9.0 Hz, 2H), 6.59 (dd, J 9.0, 5.8
Hz, 2H), 6.22 (dd, J 31.1, 7.5 Hz, 1H), 5.44 (dd, J 7.5, 4.2 Hz, 1H),
5.23−5.29 (m, 1H), 3.72−4.41 (m, 1H), 3.61−3.69 (m, 9H), 3.35−
3.52 (m, 1H), 3.09−3.25 (m, 1H), 2.60−2.86 (m, 10H), 2.48 (s, 4H),
2.07 (t, J 2.9 Hz, 3H), 1.03−1.32 (m, 15H); 13C NMR (75 MHz,
DMSO-d6) δ 207.1, 207.0, 182.2, 172.3, 172.2, 166.2, 160.7, 158.84,
158.76, 158.5, 145.3, 145.2, 135.8, 135.5, 135.2, 133.8, 132.9, 130.6,
130.0, 129.9, 129.0, 128.9, 127.94, 127.92, 119.5, 119.3, 113.4, 113.3,
86.8, 82.6, 73.4, 72.8, 63.9, 60.9, 59.6, 58.7, 55.4, 48.9, 47.3, 43.1, 38.7,
37.7, 30.0, 29.8, 27.7, 24.9, 24.6; 31P NMR (81 MHz, DMSO-d6) δ
151.5, 149.0; HRMS (ESI Q-TOF) calcd for C52H59N7O10P (M + H)+
972.4061, found 972.4029.
1
solid: H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.67 (s, 1H),
Synthesis of the Branchpoint Monomer (Z) for Method Z
(Scheme 1C). N6-Benzoyl-3′,5′-O-[1,1,3,3-tetrakis(1-methylethyl)-
1,3-disiloxanediyl]-adenosine-9-β-D-arabinofuranoside (10). Title
compound was synthesized according to previously published
protocols.33
8.35 (s, 1H), 8.03 (d, J 7.3 Hz, 2H), 7.63 (t, J 7.4 Hz, 1H), 7.53 (t, J
7.6 Hz, 2H), 7.35−7.42 (m, 2H), 7.15−7.27 (m, 7H), 6.76−6.89 (m,
4H), 6.63 (d, J 5.9 Hz, 1H), 5.93 (d, J 5.6 Hz, 1H), 5.31 (t, J 5.8 Hz,
1H), 4.52 (dd, J 12.0, 5.9 Hz, 1H), 4.11 (td, J 6.9, 3.1 Hz, 1H), 3.70 (t,
J 4.4 Hz, 6H), 3.42 (dd, J 10.3, 7.3 Hz, 1H), 3.31−3.35 (m, 1H), 3.27
(dd, J 10.3, 2.9, 1H), 2.31 (t, J 6.9 Hz, 2H), 2.17 (dt, J 16.9, 6.8 Hz,
1H), 1.87−1.98 (m, 4H); 13C NMR (75 MHz, DMSO- d6) δ 206.5,
171.6, 166.0, 158.5, 152.3, 152.2, 150.8, 145.2, 143.7, 136.0, 135.8,
133.8, 132.9, 130.2, 128.9, 128.2, 128.1, 127.1, 125.6, 113.6, 85.9, 82.0,
81.7, 78.0, 73.2, 63.9, 55.4, 37.4, 29.7, 27.6; HRMS (ESI Q-TOF)
calcd for C43H41N5O9Na (M + Na)+ 794.2802, found 794.2832.
N6-Benzoyl-2′-O-(4,4′-dimethoxytrityl)-3′-O-[(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite]-5′-O-levulinyl-adenosine-9-β-D-ara-
binofuranoside (14). Compound 13 (3.19 g. 4.13 mmol) was
dissolved in 25 mL THF, and the mixture was stirred at room
temperature under Ar. N,N-Diisopropylethylamine (5.2 mL, 32 mmol)
was added, followed by 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite (0.97 mL, 4.3 mmol). After 2 h, CH2Cl2 and NaHCO3 (sat
aq) were added, and the mixture was separated. The organic phase was
washed twice with NaHCO3 and once with brine. The combined
aqueous layers were extracted 3 times with CH2Cl2, and the combined
organic extracts were washed once with brine and subsequently
combined with the original organic layer, dried with MgSO4, filtered,
and concentrated. The residue was purified by column chromatog-
raphy (SiO2, CH2Cl2 + 1% NEt3) to yield compound 9 (3.78 g, 97%)
as an amorphous white solid: 1H NMR (400 MHz, DMSO-d6) δ 11.22
(s, 1H), 8.62 (s, 1H), 8.40 (d, J 13.7 Hz, 1H), 8.03 (d, J 7.9 Hz, 2H),
7.63 (t, J 7.3 Hz, 1H), 7.54 (t, J 7.6 Hz, 2H), 7.32−7.42 (m, 3H),
7.12−7.29 (m, 6H), 6.74−6.86 (m, 4H), 6.68 (dd, J 8.1, 6.1 Hz, 1H),
5.52 (dt, J 30.6, 5.8 Hz, 1H), 4.83−4.97 (m, 1H), 4.16−4.35 (m, 1H),
3.66−3.80 (m, 7H), 3.35−3.64 (m, 4H), 3.31 (d, J 7.1 Hz, 2H), 2.73
(t, J 5.9 Hz, 1H), 2.62 (t, J 5.9 Hz, 1H), 2.44−2.54 (m, 2H), 2.24−
2.37 (m, 2H), 2.07−2.23 (m, 1H), 1.83−2.00 (m, 3H), 1.01−1.35 (m,
10H), 0.91 (dd, J 24.7, 7.0 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ
207.1, 206.9, 172.4, 172.2, 166.1, 158.8, 158.6, 158.5, 152.4, 152.3,
151.5, 150.9, 150.9, 145.3, 145.2, 144.9, 144.5, 135.8, 135.6, 135.3,
135.2, 133.8, 132.9, 130.6, 130.0, 129.9, 129.0, 128.9, 128.7, 128.1,
127.9, 127.2, 126.9, 126.7, 119.4, 119.3, 113.5, 113.3, 87.2, 86.8, 86.7,
82.3, 81.3, 73.4, 72.7, 63.8, 63.4, 60.7, 59.7, 59.5, 58.2, 58.0, 55.5, 55.4,
55.3, 52.4, 43.5, 43.3, 43.1, 42.9, 38.7, 37.7, 37.7, 29.9, 27.8, 27.7, 25.1,
25.0, 24.8, 24.5, 24.4, 20.5, 20.4, 20.2, 20.1, 7.6; 31P NMR (81 MHz,
N6-Benzoyl-2′-O-levulinyl-3′,5′-O-[1,1,3,3-tetrakis(1-methyleth-
yl)-1,3-disiloxanediyl]-adenosine-9-β-D-arabinofuranoside (11).
N,N′-Dicyclohexylcarbodiimide (3.35 g, 16.2 mmol) was dissolved in
40 mL of anhydrous THF. Freshly distilled levulinic acid (3.3 mL, 33
mmol) was added to the mixture yielding almost immediate
precipitation. After 2 h, the mixture was filtered under nitrogen
through an oven-dried filtration apparatus designated for anhydrous
filtration. This yielded a 0.41 M solution of levulinic anhydride in
THF. Compound 10 (7.21 g, 11.7 mmol) and 4-dimethylaminopyr-
idine (845 mg, 6.91 mmol) were dissolved in THF (55.4 mL), N,N-
diisopropylethylamine (17 mL, 20 mmol) was added, and the mixture
was stirred at room temperature under Ar. The filtered levulinic
anhydride solution (31.4 mL, 12.9 mmol) was added. After 3 h,
CH2Cl2 and NaHCO3 (sat aq) were added, and the mixture was
separated. The organic phase was washed twice with NaHCO3 (sat aq)
and once with brine. The combined aqueous layers were extracted 3
times with CH2Cl2. Next the combined organic extracts were washed
once with brine and subsequently combined with the original organic
layer, dried with MgSO4, filtered, and concentrated. The residue was
purified by column chromatography (SiO2, 1:200 → 1:20 CH3OH/
CH2Cl2) to yield compound 11 (6.21 g, 74%) as an amorphous white
solid: 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.63−8.70 (m,
1H), 8.35−8.42 (m, 1H), 8.02 (dd, J 5.2, 3.3 Hz, 2H), 7.56−7.68 (m,
1H), 7.47−7.56 (m, 2H), 6.56 (d, J 7.0 Hz, 1H), 5.60 (dd, J 8.0, 7.0
Hz, 1H), 5.11 (t, J 8.3 Hz, 1H), 4.14−4.24 (m, 1H), 3.97 (ddd, J 13.7,
8.0, 3.0 Hz, 2H), 2.20−2.36 (m, 2H), 2.05−2.18 (m, 1H), 1.89 (d, J
3.1 Hz, 3H), 1.83 (dt, J 17.0, 6.3 Hz, 1H), 1.20−1.30 (m, 1H), 1.16−
1.09 (m, 6H), 0.99−1.06 (m, 20H), 0.94 (ddd, J 9.6, 8.7, 4.6 Hz, 1H);
13C NMR (75 MHz, DMSO-d6) δ 206.1, 171.6, 166.1, 152.3, 151.0,
143.8, 133.8, 132.9, 131.6, 128.94, 128.89, 128.6, 127.9, 125.9, 80.8,
79.6, 77.5, 73.7, 62.0, 37.2, 29.6, 27.4, 17.8, 17.7, 17.63, 17.61, 17.3,
17.25, 17.23, 17.1, 13.1, 12.9, 12.7, 12.4; HRMS (ESI Q-TOF) m/z
calcd for C34H50N5O8Si2 (M + H)+ 712.3198, found 712.3184.
N6-Benzoyl-2′-O-levulinyl-adenosine-9-β-D-arabinofuranoside
(12). Compound 11 (4.79 mg, 6.72 mmol) was dissolved in THF
(16.5 mL) and stirred at room temperature under Ar. Subsequently
TREAT-HF (2.0 mL, 12 mmol) was added, and the reaction was
974
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DMSO-d6) δ 149.55, 149.30, 13.86; HRMS (ESI Q-TOF) calcd for
(21) Agback, P.; Glemarec, C.; Yin, L.; Sandstrom, A.; Plavec, J.;
Sund, C.; Yamakage, S.; Viswanadham, G.; Rousse, B.; Puri, N.;
Chattopadhyaya, J. Tetrahedron Lett. 1993, 34, 3929.
(22) Damha, M. J.; Ganeshan, K.; Hudson, R. H. E.; Zabarylo, S. V.
Nucleic Acids Res. 1992, 20, 6565.
C52H59N7O10P (M + H)+ 972.4061, found 972.4088.
ASSOCIATED CONTENT
■
S
* Supporting Information
(23) Hudson, R. H. E.; Damha, M. J. J. Am. Chem. Soc. 1993, 115,
Additional data for molecules listed in Table 1. These include
HPLC chromatograms and PAGE gels on crude bRNA
products, debranching assays, inhibition studies. Additionally,
optimization parameters are listed in Figure S2. Finally,
characterization raw data are provided, including mass
spectrometry of oligonucleotides, 1H, 13C, 31P NMR and
HRMS of branchpoint amidites and intermediates. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2119.
(24) Ganeshan, K.; Tadey, T.; Nam, K.; Braich, R.; Purdy, W. C.;
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(25) Sproat, B. S.; Beijer, B.; Grotli, M.; Ryder, U.; Morand, K. L.;
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495.
(27) Grotli, M.; Eritja, R.; Sproat, B. Tetrahedron 1997, 53, 11317.
(28) Vonburen, M.; Petersen, G. V.; Rasmussen, K.; Brandenburg,
G.; Wengel, J.; Kirpekar, F. Tetrahedron 1995, 51, 8491.
(29) Brandenburg, G.; Petersen, G. V.; Rasmussen, K.; Wengel, J.
Bioorg. Med. Chem. Lett. 1995, 5, 791.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: masad.damha@mcgill.ca.
■
Notes
The authors declare no competing financial interest.
(33) Kwiatkowski, M.; Gioeli, C.; Oberg, B.; Chattopadhyaya, J. B.
Chem. Scr. 1981, 18, 95.
(34) Zavgorodny, S.; Polianski, M.; Besidsky, E.; Kriukov, V.; Sanin,
A.; Pokrovskaya, M.; Gurskaya, G.; Lonnberg, H.; Azhayev, A.
Tetrahedron Lett. 1991, 32, 7593.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Science
and Engineering Council of Canada (Discovery Grant to
M.J.D.), the Robert A. Welch Foundation (AQ-1399) (P.J.H.),
the National Science Foundation (DBI-0905865) (E.M.), and
the Swedish Research Council (R.J.).
(35) Lackey, J. G.; Mitra, D.; Somoza, M. M.; Cerrina, F.; Damha, M.
J. J. Am. Chem. Soc. 2009, 131, 8496.
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Xu, R. M.; Damha, M. J.; Ron, D. RNA 2008, 14, 225.
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