N2-Modified 2-aminopurine ribonucleosides as minor-groove-modulating adenosine replacements in duplex RNA

Article abstract of DOI:10.1021/ol100019r

"Chemical equation presented" Nucleoside analogs that project substituents Into the minor groove when incorporated into duplex RNA perturb the binding of proteins and can affect base pairing specificity. The synthesis of 2-amlnopurine ribonucleoside analo

Full text of DOI:10.1021/ol100019r

ORGANIC
LETTERS
N2-Modified 2-aminopurine
ribonucleosides as
2010
Vol. 12, No. 5
1044-1047
minor-groove-modulating adenosine
replacements in duplex RNA
Hayden Peacock, Olena Maydanovych, and Peter A. Beal*,*
Department of Chemistry, UniVersity of California, DaVis, California 95616
beal@chem.ucdaVis.edu
Received January 7, 2010
ABSTRACT
Nucleoside analogs that project substituents into the minor groove when incorporated into duplex RNA perturb the binding of proteins and
can affect base pairing specificity. The synthesis of 2-aminopurine ribonucleoside analogs and their phosphoramidites, their incorporation
into duplex RNA, their postsynthetic modification via Cu-catalyzed azide-alkyne cycloaddition (CuAAC), and their effect on duplex stability
and base pairing specificity are described.
Nucleoside analogs incorporated into RNA have a variety
of uses such as investigating reaction mechanisms involving
RNA,1,2 imparting favorable properties on siRNAs,3,4 prob-
ing RNA structure and function5,6 and exploring interaction
between RNAs and proteins or small molecules.7 In our
previous studies, we showed that site-specific incorporation
of the nucleoside analogs N2-benzylguanosine or N2-benzyl-
2′-deoxyguanosine into duplex RNA blocked the binding of
dsRBMs (double-stranded RNA binding motifs, 65-70
amino acid sequence motifs found in many cellular proteins
that bind duplex RNA8-11). Since dsRBMs bind RNA
through minor groove contacts,12,13 and the 2-amino group
of guanosine is directed into the minor groove, the introduc-
tion of the N2-benzyl analogs at specific sites controlled the
binding of the dsRBM-containing proteins. This allowed us
to characterize the binding of the RNA-dependent protein
kinase (PKR) to siRNA9 and the binding of the RNA editing
enzyme ADAR2 to model substrates.10 However, since these
early studies only employed N2-benzylguanosine as the
modified base, the approach was limited to the modification
of G•C pairs and to a single N2 substituent. Therefore, as an
extension of this work, it was desirable to develop new
analogs that would allow for the modification of the RNA
minor groove at A•U pairs and with a variety substituents.
In addition, nucleoside analogs that block dsRBM binding
* Phone: (530) 7524132. Fax: (530) 752-8995.
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10.1021/ol100019r  2010 American Chemical Society
Published on Web 01/28/2010

may improve siRNA performance, since dsRBM proteins
such as PKR and the ADARs can impede siRNAs from
causing the desired specific RNAi effect.14-17 New ways of
directing functionality into the minor groove of duplex RNA
will therefore also be of interest in siRNA design.
faster with fluoride leaving groups than other halide leaving
groups,20 we prepared a 2-fluoropurine ribonucleoside pre-
cursor to evaluate it as a substrate for substitution reactions
with various amines (Scheme 1). For the purposes of
comparison, we also investigated a 2-bromopurine ribo-
nucleoside intermediate as an alternative precursor to 2-ami-
nopurine derivatives.
To create new nucleoside analogs that could be used to
modify A•U pairs in RNA with minor groove substituents,
we chose to synthesize derivatives of 2-aminopurine ribo-
nucleoside bearing different substituents at N2 (Figure 1).
Like guanosine, 2-aminopurine in duplex RNA projects the
2-amino group into the minor groove. Furthermore, 2-ami-
nopurine is an effective replacement for adenosine as it is
anticipated to form a stable, two hydrogen bond pair with
uridine. However, there are no reports on the effect of N2-
modified 2-aminopurine ribonucleoside substitution for ad-
enosine in duplex RNA. Here we describe the synthesis of
2-aminopurine ribonucleoside analogs and their phosphora-
midites, their incorporation into RNA, their postsynthetic
modification via Cu-catalyzed azide-alkyne cycloaddition
(CuAAC) and their effect on duplex stability and base pairing
specificity.
We chose to protect the ribose hydroxyl groups of the
2-fluoropurine ribonucleoside intermediate in acetate esters
to allow for convenient deprotection at the amine substitution
step. Thus, 2′,3′,5′-tri-O-acetyl-2-fluoropurine ribonucleoside
(3) was prepared by dehalo hydrogenolysis and diazotization/
fluorination of 2′,3′,5′-tri-O-acetyl-6-chloro-2-aminopurine
(1). The 6-chloro hydrogenolysis substrate was readily
prepared from guanosine via the method of Robins and
Uznanski.21 Hydrogenolysis of 1 has previously been
reported in methanol;22 however, we found the reaction was
more efficient upon addition of 10% THF to the solvent,
delivering 2′,3′,5′-tri-O-acetyl-2-aminopurine (2) in 84%
yield. Diazotization/fluorination of 2 proceeded in excellent
yield to afford the acetyl protected 2-fluoropurine intermedi-
ate. We evaluated the 2-fluoropurine intermediate as a
substrate for SNAr reactions by treating it with amines in
DMF or THF. Cyclopentyl and propyl groups were chosen
for incorporation at N2 in order to investigate the effect of
varying the size of the minor groove obstruction in an RNA
duplex on duplex stability and protein binding. To prepare
RNA amenable to postsynthetic modification via CuAAC
reaction, we also introduced a propargyl group. For the
cyclopentylamine and propylamine substitutions, the substi-
tution reactions occurred rapidly at ambient temperature with
an excess of alkylamine in DMF, consuming the fluoropurine
starting material in less than 30 min. After completion of
the substitution reaction, it was necessary to add NH3/
methanol to the reaction mixture to achieve complete acetyl
deprotection. Chromatographic purification yielded free
nucleoside derivatives 4 and 5 in good yield. The rate of
substitution with propargylamine was slightly slower. After
3 h in THF at 65 °C, the fluoropurine was completely
consumed. Subsequent NH3/methanol treatment and purifica-
tion afforded the desired N2-modified 2-aminopurine ribo-
nucleoside derivative 6 in good yield.
Figure 1. (A) Structure of an A•U base pair in duplex RNA,
showing location of adenosine C2.18 (B) N2-Modified 2-aminopu-
rines place steric bulk (R) in the duplex RNA minor groove.
The SNAr reaction with amines was also investigated using
2′,3′,5′-tri-O-tert-butyldimethylsilyl-2-bromopurine ribonu-
cleoside (8) as a substrate. The bromopurine intermediate
was prepared by diazotization/bromination23 of tri-O-TB-
DMS protected 2-aminopurine ribonucleoside, which was
generated in one step from commercially available 6-mer-
capto-2-aminopurine ribonucleoside.24 SNAr reactions with
excess amine occurred in 1-5 h at 75-80 °C in DMF in
good yields to afford the tri-O-TBDMS N2-modified ribo-
nucleosides 9-14 (Scheme 2). Deprotection to the free
nucleoside was demonstrated on the TBDMS-protected N2-
The preparation of the analog N2, N2-dimethyl-2-aminopu-
rine ribonucleoside has previously been reported via SNAr
reaction between a 2-chloropurine ribonucleoside precursor
and dimethylamine.19 Since SNAr reactions typically proceed
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Oxford, 2009; Vol. 1175, pp 40-54.
(20) Liu, J.; Robins, M. J. J. Am. Chem. Soc. 2007, 129, 5962–8.
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Bear, J. S.; Gough, G. R.; Gilham, P. T.; Moras, D. J. Mol. Biol. 1989,
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Org. Lett., Vol. 12, No. 5, 2010
1045

Scheme 1
.
Preparation of N2-Modified 2-Aminopurine
Scheme 3. Preparation of Phosphoramidites
Ribonucleosides via 2-Fluoropurine Intermediate
to impart favorable delivery properties on siRNAs. We chose
to employ the water-soluble tris-(hydroxypropyltriazolylm-
ethyl)amine CuI ligand, which has previously been reported
in the CuAAC reaction with oligonucleotide substrates.28,29
Its solubility offers the advantage of completely aqueous
reaction media, which will be beneficial in future applications
where native RNA structure must be maintained during the
reaction. We prepared the ligand from tripropargylamine and
3-azido-propanol via CuBr catalysis, as detailed in the
Supporting Information. Click products of the 12-mer RNA
were generated by incubating an aqueous solution of the
single-stranded RNA with tris-(hydroxypropyltriazolylm-
ethyl)amine, CuSO4, sodium ascorbate and azide (5 equiv)
for 4 h at ambient temperature. The click products were
purified from the reaction mixture by denaturing polyacry-
lamide gel electrophoresis (PAGE) and confirmed by ESI-
MS. CuAAc reaction with N-azidoacetyl-D-mannosamine30
(ManNAc-N3) went to completion, as determined by a
quantitative shift in mobility of the RNA band on PAGE.
However, the reaction of the same RNA with 2-azido-2-
deoxy-D-mannose30 (Man-2-N3) and AZT, both secondary
azides, went to approximately 50% completion. In each case,
the click product was purified by extraction of the slower
gel band. The lower conversion of alkynyl RNA in the
presence of these two azides might be due to the relatively
short length of the alkynyl linker. Incomplete conversion of
alkyne-containing oligonucleotides via CuAAC reactions has
been previously been observed with short alkynyl linkers
connected to nucleobases in single-stranded DNA.31 The
ManNAc click product was formed from the 21-mer via the
same protocol. The reaction consumed all alkynyl RNA and
yielded approximately equal amounts of mono- and diclick
products, which were readily separated by PAGE.
benzyl derivative 11 by treatment with TBAF, isolating the
free nucleoside 15. Both the tri-O-acetyl fluoropurine inter-
mediate and the tri-O-TBDMS bromopurine intermediate
gave acceptable yields in convenient reaction times. How-
ever, retention of ribose protection by the tri-O-TBDMS
substrate allows for further elaboration of the nucleoside
structure.
Scheme 2.
Preparation of N2-Modified 2-Aminopurine
Ribonucleosides via 2-Bromopurine Intermediate
The effect of the N2-amino substitutions on duplex stability
was investigated via thermal denaturation (Tm) studies of a
12 base pair RNA duplex (Figure 2). The nucleoside analogs
were incorporated into the duplex opposite each of the four
Nucleosides 4-6 were converted to 5′-O-DMTr, 2′-O-
TBDMS, ꢀ-cyanoethyl phosphoramidites 22-24 following
conventional procedures, and incorporated into a 12-mer
RNA by solid-phase synthesis (Scheme 3). The N2-propargyl
amidite 24 was also incorporated at two positions in a 21-
mer RNA which forms the sense strand of an siRNA
targeting the human caspase 2 message.
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The RNAs incorporating N2-propargyl-2-aminopurine were
further modified via CuAAC reaction25-27 with three azides
of interest (Scheme 4). The azides were chosen based on
their potential to deliver steric bulk to the minor groove and
(30) Chokhawala, H. A.; Huang, S. S.; Lau, K.; Yu, H.; Cheng, J. S.;
Thon, V.; Hurtado-Ziola, N.; Guerrero, J. A.; Varki, A.; Chen, X. ACS
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1046
Org. Lett., Vol. 12, No. 5, 2010

Scheme 4. Post-synthetic RNA modification via CuAAC
Figure 2. Thermal denaturation (Tm) analysis of 12-mer RNA
duplexes containing adenosine, 2-aminopurine, and N2-modified
2-aminopurine bases opposite the four common nucleobases.
common bases (adenine (A), guanine (G), cytosine (C), and
uracil (U)). 2-Aminopurine was also incorporated opposite
each of the common bases for comparison. First, the Tm of
each of the modifications opposite U was examined to
determine the ability of the modifications to replace A in an
A•U base pair (gray bars). The duplex containing an A•U
base pair had a measured Tm of 40.7 ( 0.8 °C. Replacement
of A with the N2-cyclopentyl and N2-propyl 2-aminopurine
analogs resulted in a small destabilization, within 1.3 ( 0.8
°C of the natural base. The Tm analysis also indicated that
two of the bulky triazole-containing click products, ManNAc
and AZT, were well-accommodated in the minor groove,
both within 1.3 ( 0.8 °C of the natural A•U pair. Therefore,
these four analogs are excellent replacements of A in duplex
RNA. Furthermore, they project substituents with a range
of size and structure in to the minor groove and can therefore
be used to study minor groove contacts at A•U sites and to
disrupt complexes with dsRBMs. The greatest destabilization
opposite U was presented by the N2-propargyl modification,
at 4.5 ( 0.7 °C, for reasons yet to be defined.
The change in Tm of each of the modifications in
mismatches against C, A and G was next examined to
determine the effect of the modifications on pairing specific-
ity (Figure 2, blue, black and red bars, respectively). A
substantial effect on specificity was presented by the N2-
cyclopentyl modification. This modification causes a 5.1 (
0.6 °C destabilization when placed opposite C relative to
the natural A•C mismatch, but its stability opposite U was
within 1.3 ( 0.8 °C of the A•U base pair. Therefore, this
new modified nucleoside is more specific for U than natural
A in the context of the 12-mer RNA studied. This increased
specificity profile of the N2-cyclopentyl modification is
important since its incorporation into the antisense strand of
an siRNA could make the siRNA more specific for its
intended target, generating RNAi reagents with fewer off-
target effects. Experiments to explore this effect are currently
underway in our laboratory. The three click products showed
similar specificity profiles to A. Given the similar base
pairing stability and specificity of the ManNAc click product
compared to A and the substantial steric bulk this analog
introduces into the RNA minor groove, it was chosen for
incorporation into siRNA as detailed above for future studies
on protein binding and RNAi activity.
In summary, N2-modified 2-aminopurine analogs can be
generated in good yield via SNAr reaction between amines
and 2-fluoro and 2-bromo precursors, readily incorporated
into duplex RNA and modified postsynthesis via CuAAC
reaction with azides. Four of six modifications were well-
tolerated as adenosine replacements, including two excep-
tionally bulky triazole-containing click products. The base
pairing specificity profile was also affected by the minor
groove projections, with the N2-cyclopentyl derivative show-
ing increased specificity for uracil relative to adenine. Results
of protein binding and RNAi studies with RNAs incorporat-
ing these new nucleoside analogs will be reported in due
course.
Acknowledgment. We thank Professor Xi Chen, Dr.
Hongzhi Cao and Saddam Muthana (Department of Chem-
istry, University of California, Davis) for gifts of 2-azido-
2-deoxy-D-mannose and N-azidoacetyl-D-mannosamine. P.A.B.
acknowledges support from the National Institutes of Health
(R01 GM080784).
Supporting Information Available: Synthetic procedures
and characterization data for 2-6 and 8-24 and Cu ligand
used in CuAAC; procedures concerning RNA preparation,
CuAAC reaction, duplex formation and Tm analysis; Table
of Tm data. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL100019R
Org. Lett., Vol. 12, No. 5, 2010
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