Synthesis and characterization of oligodeoxyribonucleotides modified with 2′-amino-α-l-LNA adenine monomers: High-affinity targeting of single-stranded DNA

Article abstract of DOI:10.1021/jo4022937

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diaster

Full text of DOI:10.1021/jo4022937

Article
pubs.acs.org/joc
Synthesis and Characterization of Oligodeoxyribonucleotides
Modified with 2′-Amino-α‑L‑LNA Adenine Monomers: High-Affinity
Targeting of Single-Stranded DNA
Nicolai K. Andersen,†,§ Brooke A. Anderson,‡,§ Jesper Wengel,† and Patrick J. Hrdlicka‡,*
†Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense, Denmark
‡Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343, United States
S
* Supporting Information
ABSTRACT: The development of conformationally restricted nucleotide building blocks continues to attract considerable
interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA)
and its diastereomer α-L-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly
increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to
unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs)
modified with 2′-amino-α-L-LNA adenine monomers W−Z. The synthesis of the target phosphoramidites 1−4 is initiated from
pentafuranose 5, which upon Vorbruggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields
̈
nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine
intermediate 9, which after a series of nontrivial protecting-group manipulations affords key intermediate 15. Subsequent
chemoselective N2′-functionalization and O3′-phosphitylation give targets 1−4 in ∼1−3% overall yield over 11 steps from 5.
ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X−Z display greatly increased affinity toward
DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex
stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNAs of
considerable interest for DNA-targeting applications.
including very high affinity toward DNA/RNA targets, but is
less well characterized as a result of its more limited commercial
availability.6 The interesting properties of LNA, α-L-LNA, and
other conformationally restricted nucleotides have spurred the
development of many analogues.1,7
As part of our ongoing interest in LNA chemistry and
diagnostic applications of pyrene-functionalized oligonucleoti-
des,2c,8 we recently pursued the development of N2′-pyrene-
functionalized 2′-amino-α-L-LNA thymine monomers (Figure
1).9 Oligodeoxyribonucleotides (ONs) modified with these
monomers display remarkable affinity toward complementary
DNA, as the pyrene moieties are preorganized to intercalate
and engage in stacking with neighboring nucleobases upon
INTRODUCTION
■
Major efforts have been devoted over the past 20 years to the
development of conformationally restricted nucleotides.
Oligonucleotides that are modified with these building blocks
often display markedly increased affinity toward nucleic acid
targets, improved discrimination of nontargets, and greater
resistance against enzymatic degradation relative to reference
strands.1 Such oligonucleotides are accordingly widely used for
nucleic acid-targeting applications in molecular biology,
biotechnology, and medicinal chemistry.2 Locked nucleic acid
(LNA) is one of the most prominent members of this
compound class because of its extraordinary affinity toward
DNA and RNA complements (Figure 1); thus, increases in the
thermal denaturation temperature (Tm) of up to +10 °C per
modification have been observed.3−5 The diastereomeric α-L-
LNA (Figure 1) shares many characteristics with LNA,
Received: October 13, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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Figure 1. Structures of LNA, α-L-LNA, and the 2′-amino-α-L-LNA monomers studied herein. T = thymin-1-yl.
a
Scheme 1. Retrosynthetic Analysis of 2′-Amino-α-L-LNA-adenine Monomers W−Z
a
Abbreviations: PG = PN(iPr)2OCH2CH2CN; R = H, CH2Py, COPy, and COCH2Py for W−Z, respectively; py = pyren-1-yl; ABz = 6-N-
benzoyladenin-9-yl.
duplex formation.9b Thus, increases in Tm of up to 20 °C per
modification have been observed for short ONs modified with
these building blocks. We have taken advantage of these
characteristics and have developed probes for a variety of
diagnostic applications. For example, ONs modified with two
next-nearest-neighbor incorporations of 2′-N-(pyren-1-yl)-
acetyl-2′-amino-α-L-LNA-T monomers are promising tools for
the detection of single-nucleotide polymorphisms, which are
the most prevalent type of genetic mutation in the human
genome.10 These probes efficiently discriminate between
complementary and single-base-mismatched DNA/RNA tar-
gets through differences in pyrene excimer emission levels. In
another example, the fluorescence of ONs modified with 2′-N-
(pyren-1-yl)acetyl-2′-amino-α-L-LNA-T monomers was found
to increase 16-fold upon hybridization with DNA targets
featuring abasic sites (i.e., DNA lesions that result in genomic
mutations and emergence of cancers if unrepaired).11 We have
also utilized N2′-pyrene-functionalized 2′-amino-α-L-LNA-T
monomers as the key activating components of the so-called
Invader probes, which recognize mixed-sequence double-
stranded DNA.12 Unfortunately, the synthesis of N2′-pyrene-
functionalized 2′-amino-α-L-LNA-thymine phosphoramidites is
challenging (∼20 steps, <4% overall yield from diacetone-α-D-
glucose), mainly because of unsuccessful attempts to introduce
the necessary C2′-azido group at the nucleoside level without
concomitant O2,O2′-anhydronucleoside formation.9a This
forced us to introduce the azido group at the carbohydrate
level, which resulted in a loss of anchimeric assistance from the
O2-position during Vorbruggen glycosylation and the for-
̈
mation of anomeric nucleoside mixtures. We hypothesized that
these synthetic difficulties might be overcome with the
corresponding adenine derivates, as the formation of
anhydronucleosides would be unlikely. Easier access to N2′-
pyrene-functionalized 2′-amino-α-L-LNA monomers is desir-
able in order to evaluate the diagnostic potential of these
building blocks in greater detail.
In the present article, we report the synthesis and
characterization of ONs modified with four different 2′-
amino-α-L-LNA-adenine monomers W−Z (Figure 1). The
ONs were characterized via thermal denaturation, UV−vis, and
fluorescence experiments and shown to display extraordinary
thermal affinity toward complementary DNA (ΔTm/modifica-
tion up to 14.0 °C) and photophysical characteristics consistent
with intercalative binding modes for the pyrene moieties.13
RESULTS AND DISCUSSION
■
Retrosynthetic Analysis of 2′-Amino-α-L-LNA-A
Monomers. Inspired by our previously reported synthesis of
the corresponding thymine monomers,9 we identified adenine
derivative 15 as a suitable substrate for chemoselective N2′-
functionalization and subsequent O3′-phosphitylation, which
were expected to yield the target 6-N-benzoyladenin-9-yl
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a
Scheme 2. Synthesis of Intermediate 9
a
Abbreviations: BSA = N,O-bis(trimethylsilyl)acetamide; 1,2-DCE = 1,2-dichloroethane; ABz = 6-N-benzoyladenin-9-yl; GNO3 = guanidinium
nitrate; 15-C-5: 15-crown-5.
a
Scheme 3. Synthesis of Key Intermediate 15
a
Abbreviations: ABz = 6-N-benzoyladenin-9-yl; DMTr = 4,4′-dimethoxytrityl; FmocCl = 9′-fluorenylmethyl chloroformate.
nucleosides 1−4 (Scheme 1). We surmised that key
intermediate 15 could be obtained from nucleoside 9 via a
series of protecting-group manipulations. The O3′-benzyl
group was selected because of (i) its stability under acidic
and basic reaction conditions, allowing it to be introduced at
the beginning of the synthetic route, and (ii) its ability to be
removed under conditions that have only a minimal effect on
the 6-N-benzoyl group of adenine.14 We expected to construct
the 2-oxo-5-azabicyclo[2.2.1]heptane skeleton of 9 in a manner
similar to that originally reported for 2′-amino-β-D-LNA,15
namely, via a one-pot tandem Staudinger/intramolecular
nucleophilic substitution reaction, which revealed nucleoside
8 as an early target. In contrast to our synthesis of the
corresponding thymine monomer, where the C2′-azido group
had to be introduced at the carbohydrate stage,9a we decided to
introduce the 2′-azido group of 8 at the nucleoside stage,
anticipating that selective O2′-deacylation of known nucleoside
66b and subsequent O2′-activation and introduction of the
azido group would provide 8. Importantly, the anchimeric
assistance provided by the O2-substituent during the
Vorbruggen glycosylation of 5 to give 6 prevents formation
̈
of anomeric mixtures, which is one of the drawbacks in the
synthesis of 2′-amino-α-L-LNA-T monomers.9a
Synthesis of Key Intermediate 15. Fully protected
pentafuranose 5, which was obtained from diacetone-α-D-
glucose in six steps and ∼30% overall yield,6b,16 served as the
starting material for the synthesis of key intermediate 15
(Scheme 2). Glycosyl donor 5 was converted into alcohol 7
following protocols that deviated from the known route6b in the
following manner: (i) the use of trimethylsilyl triflate as the
Lewis acid and 1,2-dichloroethane as the reaction solvent
during Vorbruggen glycosylation of 5 afforded 6 in higher yield
̈
and with easier workup than the original protocol involving
tin(IV) chloride and acetonitrile (70% vs 57%, respectively);
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(ii) the use of the guanidinium nitrate/sodium methoxide
reagent mixture17 resulted in slightly more efficient O2′-
deacylation of 6 than the original protocol enploying dilute
methanolic ammonia (88% vs 79% yield), although the dilute
reaction conditions of the former approach rendered it less
practical for large-scale reactions. Alcohol 7 was O2′-triflated
and treated with sodium azide and 15-crown-5 in DMF at
elevated temperatures to furnish azide 8 in excellent yield (89%
over two steps). IR spectra of 8 verified the presence of the
azide group (sharp band at 2115 cm−1). Nucleoside 8 was
converted into the desired 2′-amino-α-L-LNA intermediate 9
via a one-pot tandem Staudinger/intramolecular nucleophilic
substitution reaction using alkaline trimethylphosphine15 in a
satisfying 85% yield.18 The 2-oxo-5-azabicyclo[2.2.1]heptane
skeleton and stereochemical configuration of 9 were verified by
NOE difference experiments on downstream products (vide
infra).
Structural Verification of the 2′-Amino-α-L-LNA
1
Configuration. In agreement with previously reported H
NMR signals of other α-L-LNA nucleosides,6b,9 the H NMR
1
signals of H1′, H2′, and H3′ of 2′-amino-α-L-LNA nucleosides
appear as singlets or narrow doublets (J < 2 Hz)20 since the
H1′−C1′−C2′−H2′ and H2′−C2′−C3′−H3′ torsion angles
are fixed in +gauche and −gauche conformations, respectively.
The structure of bicyclic nucleoside 14 was ascertained by NOE
difference spectroscopy. NOE contacts between H1′ and H2′
(7%), H1′ and H3′ (5%), and H2′ and H3′ (2%) suggested a
cis relationship between these protons (Figure 2). Since the
The protecting-group manipulations needed to convert
nucleoside 9 into key intermediate 15 proved to be surprisingly
challenging (Scheme 3). For example, O3′-debenzylation of
bicyclic nucleoside 9 was unsuccessful using boron trichloride
or methanesulfonic acid in dichloromethane. Similarly, the
corresponding N2′-Fmoc-protected nucleoside 10 (obtained by
reacting 9 with 9′-fluorenylmethyl chloroformate under
Schotten−Baumann conditions; results not shown) failed to
undergo O3′-debenzylation with BCl3 in dichloromethane or
by standard hydrogenolysis. Additional attempted strategies are
outlined in Scheme S1 in the Supporting Information.
Therefore, it proved necessary to protect the N2′-position of
bicyclic nucleoside 9 as a trifluoroacetamide to give nucleoside
11 in 62% yield, which was then O3′-debenzylated with BCl3 in
hexanes to give nucleoside 12 in 87% yield (Scheme 3).
Protection of the O3′-position as a napthyl ether would be an
attractive alternative option, as it can be cleaved using DDQ,7a
but this option was not considered at the time of the synthesis.
Subsequent nucleophilic substitution of the C5′-mesylate group
of 12 was attempted using potassium acetate and 18-crown-6,
but this resulted in the formation of numerous side products
according to analytical TLC, and the approach was abandoned
(Scheme 3). Instead, treatment of 12 with sodium benzoate
and 15-crown-5 in hot DMF afforded O5′-benzoylated
nucleoside 13 in 83% yield. Subsequent treatment of nucleoside
13 with aqueous sodium hydroxide in 1,4-dioxane effected the
cleavage of both the O5′-benzoyl and N2′-trifluoroacetamide
protecting groups, affording polar amino alcohol 14 in 60%
yield after column chromatography. The O5′-hydroxyl group of
nucleoside 14 was subsequently protected as the 4,4′-
dimethoxytrityl (DMTr) ether using standard conditions to
afford key intermediate 15. Curiously, the reaction proceeded
in no more than 38% yield, as significant amounts of N2′,O5′-
di-DMTr-protected nucleoside 16 were produced as well (30%
yield). Efforts to optimize the yield of 15 (e.g., slow addition of
DMTr-Cl at low temperatures) were unsuccessful. However, it
was possible to recycle nucleoside 16 into amino diol 14 using
3% dichloroacetic acid in a mixture of methanol and
nitromethane19 in up to 96% yield. An alternative strategy
toward 15, involving N2′-Fmoc protection of 14 and
subsequent O5′-DMTr-protection and N2′-Fmoc deprotection,
was dismissed because O5′-DMTr protection was inefficient
(∼40% yield; results not shown). Thus, the preferred route (5
→→ 9 → 11 →→ 15) afforded intermediate 15 in ∼5% overall
yield from 5, not taking the recycling step 16 → 14 into
account.
Figure 2. Key NOE contacts in nucleoside 14.
stereochemical configuration at C3′ is defined by the choice of
starting material and remains unchanged throughout synthesis,
H1′ and H2′ must be pointing “down”, which confirms the
nucleobase as pointing “up” and hence establishes the 2′-
amino-α-L-LNA configuration. This was substantiated by signal
enhancements between H5″A and H8′ (6%), indicating a cis
relationship between the nucleobase and H5″ (H5″A is
tentatively assigned as the H5″ closest to the nucleobase).
Synthesis of Phosphoramidite Building Blocks 1−4.
Chemoselective N2′-functionalizations of key intermediate 15
to give nucleosides 17−20 were realized as follows: (i) using 9′-
fluorenylmethyl chloroformate to give 17 in 51% yield
(Schotten−Baumann conditions could not be used because of
the low solubility of 15 in dioxane/water); (ii) via reductive
amination using 1-pyrenecarboxaldehyde and sodium triacetox-
yborohydride21 as the reducing agent to give 18 in 68% yield;
or (iii) via EDC-mediated coupling of 1-pyrenecarboxylic acid
or 1-pyreneacetic acid to give 19 in 64% yield and 20 in 79%
yield, respectively (Scheme 4). Subsequent phosphitylation
using 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite af-
forded the target compounds 1−4 in 46−71% yield after
column chromatography and precipitation.
Synthesis of Modified ONs and Experimental Design.
Phosphoramidites 1−4 were used in machine-assisted solid-
phase DNA synthesis (0.2 μmol scale) to incorporate
monomers W−Z into ONs using the following hand-coupling
conditions (activator; coupling time; stepwise coupling yield):
monomer W (pyridinium hydrochloride; 30 min; ∼82%);
monomers X−Z (pyridinium hydrochloride; 15 min; ∼95%).
Following workup and HPLC purification, the composition and
purity of all of the modified ONs were ascertained by MALDI
MS analysis (Tables S1 and S2 in the Supporting Information)
and ion-pair reversed-phase HPLC, respectively. ONs contain-
ing a single incorporation in the 5′-GTG BTA TGC context are
denoted W1, X1, Y1, and Z1, respectively. Similar conventions
apply for ONs in the B2−B6 series (see Table 1). Reference
DNA and RNA strands are denoted as D1/D2 and R1/R2,
respectively. The following descriptive nomenclature is also
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a
Scheme 4. Synthesis of Phosphoramidites 1−4
a
Abbreviations: ABz = 6-N-benzoyladenin-9-yl; DIPEA = N,N′-diisopropylethylamine; DMTr = 4,4′-dimethoxytrityl; EDC·HCl = 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; FmocCl = 9′-fluorenylmethyl chloroformate; NMI = N-methylimadazole; PCl = 2-cyanoethyl-
N,N′-diisopropylchlorophosphoramidite; Py = pyren-1-yl.
used: N2′-PyMe (X series), N2′-PyCO (Y series) and N2′-
PyAc (Z series). We previously used this 9-mer mixed-sequence
context to study the hybridization properties of ONs modified
with N2′-pyrene-functionalized 2′-amino-α-L-LNA-thymine
monomers, which facilitates a direct comparison.9
Thermal Denaturation Experiments. Thermal Affinity
toward Complementary DNA/RNA. The thermostabilities of
duplexes between W/X/Y/Z-modified ONs and complemen-
tary DNA/RNA were evaluated by determining their thermal
denaturation temperatures (Tm) in medium-salt buffer ([Na+]
= 110 mM, pH 7.0). The changes in Tm for the modified
duplexes relative to the Tm values for the unmodified reference
duplexes (ΔTm) are discussed.
ONs with one or two incorporations of 2′-amino-α-L-LNA-
adenine monomer W display very similar thermal affinities
toward complementary DNA as the unmodified reference ONs
(ΔTm between −1.0 to +0.5 °C; Table 1). Thus, unlike the
corresponding 2′-oxy-α-L-LNA-adenine monomer,6b,22 the 2-
oxo-5-azabicyclo[2.2.1]heptane skeleton of monomer W is not
inherently beneficial for DNA duplex formation. We have made
similar observations with ONs modified with the thymine
counterpart of monomer W.9a This indicates that the
unfunctionalized 2′-nitrogen of 2′-amino-α-L-LNA monomers
either stabilizes the single-stranded ON or destabilizes the
duplex, for example, by perturbing the hydration spine in the
major groove. In contrast, ONs modified with N2′-pyrene-
functionalized 2′-amino-α-L-LNA-adenine monomer X, Y, or Z
display greatly increased thermal affinity toward DNA targets
(ΔTm/modification between +2.5 and +14 °C; Table 1). The
observed Tm trends for singly modified ONs (Y > Z ≥ X)
Table 1. ΔTm Values for Duplexes between B1−B6 and
a
Complementary DNA Targets
ΔTm/°C
ON
duplex
B = W B = X B = Y
B = Z
B1
5′-GTG BTA TGC
3′-CAC TAT ACG
−0.5
+0.5
−1.0
0.0
+5.0
+7.0
+5.0
+6.5
+5.5
+5.0
+11.0
+14.0
+13.5
+11.5
+12.0
+13.5
+6.0
D2
B2
D2
5′-GTG ATB TGC
3′-CAC TAT ACG
+7.5
+16.0
+7.5
B3
D2
5′-GTG BTB TGC
3′-CAC TAT ACG
D1
B4
5′-GTG ATA TGC
3′-CAC TBT ACG
D1
B5
5′-GTG ATA TGC
3′-CAC TAT BCG
0.0
+6.0
D1
B6
5′-GTG ATA TGC
3′-CAC TBT BCG
−2.0
+14.5
a
ΔTm = change in Tm relative to the value for the unmodified
reference duplex D1:D2 (Tm ≡ 27.5 °C). Tm was determined as the
maximum of the first derivative of the melting curve (A260 vs T)
recorded in medium-salt buffer ([Na+] = 110 mM, [Cl−] = 100 mM,
pH 7.0 (NaH2PO4/Na2HPO4)) using a concentration of 1.0 μM for
each strand. Tm values are averages of at least two measurements
within 1.0 °C. A/C/G/T = adenin-9-yl/cytosin-1-yl/guanin-9-yl/
thymin-1-yl DNA monomers. For the structures of monomers W−Z,
see Figure 1. Data for the B1, B2, B4, and B5 series for monomers X,
Y, and Z were previously reported in ref 12b.
indicate that (i) monomers in which the pyrene moiety is
attached to the 2-oxo-5-azabicyclo[2.2.1]heptane skeleton via
an alkanoyl linker induce greater thermostabilization than the
corresponding monomers employing alkyl linkers (N2′-PyCO
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Y > N2′-PyMe X) and (ii) monomers with short alkanoyl
linkers result in more thermostable DNA duplexes than the
corresponding monomers with longer alkanoyl linkers (N2′-
PyCO Y > N2′-PyAc Z). We previously observed similar
structure−property relationships with the analogous thymine
monomers, which on the basis of UV−vis spectroscopy and
molecular modeling results were explained by differential
placement of the pyrene moiety for affinity-enhancing
intercalation.9b Similar structural underpinnings are likely in
effect for monomers X−Z (vide infra). However, the electron
density of the pyrene moieties, which differs among the three
monomers, may also be a contributing factor to the differential
duplex stabilization.
An interesting trend was observed for the doubly modified
ONs: incorporation of a second N2′-PyMe X or N2′-PyCO Y
monomer did not result in additionally increased thermal
affinity against DNA targets, whereas a second incorporation of
the more flexible N2′-PyAc Z monomer did (compare the ΔTm
values for the B1−B3 and B4−B6 series in Table 1). We
hypothesize that the former observation is due to violation of
the “nearest-neighbor exclusion principle”, which states that
free intercalators at most bind to every second base pair of a
DNA duplex because of limits on the local expandability of
duplexes.23 If the principle is extrapolated to tethered
intercalators and 3′-intercalative binding modes of the pyrene
moieties of monomers X−Z are assumed (vide infra), the
resulting duplexes involving B3 and B6 would feature a
localized region with two intercalators in an area defined by
four base pairs, which is at the saturation threshold. We
speculate that the greater flexibility of the N2′-PyAc Z
monomer allows the modified DNA duplexes to structurally
compensate for any stress induced by the high intercalator
density.
PyCO Y-modified duplexes being moderately stabilized (ΔTm/
modification between +1.5 and +6.5 °C; Table 2). N2′-PyAc Z-
modified ONs also displayed lower affinities toward RNA than
DNA targets but resulted in more thermostable heteroduplexes
(ΔTm/modification between +4.0 and +8.0 °C; Table 2).
These differences may again reflect the greater flexibility of
monomer Z, which allows the pyrene moieties to adopt more
favorable positions for affinity-enhancing intercalation (vide
infra). DNA-selective hybridization (Table S3 in the Support-
ing Information), as seen for X/Y/Z-modified ONs and ONs
modified with the thymine counterparts,9b is often observed for
ONs modified with intercalating moieties,24 as intercalators
favor the B-type helix geometry of DNA:DNA duplexes over
the more compressed A/B-type helix geometry of DNA:RNA
duplexes.25
Mismatch Discrimination. The binding specificities of
centrally modified ONs against DNA/RNA strands with
mismatched nucleotides opposite the W−Z monomers were
determined. W4 displayed improved discrimination of mis-
matched DNA targets relative to reference strand D2, with the
AG mismatch being discriminated particularly well (Table 3).
Table 3. Discrimination of Mismatched DNA Targets by B4
a
Series and Reference ONs
DNA: 5′-GTG ABA TGC
Tm/°C
B = T
ΔTm/°C
B = C
ON
sequence
B = A
B = G
D2
W4
X4
Y4
Z4
3′-CAC TAT ACG
3′-CAC TWT ACG
3′-CAC TXT ACG
3′-CAC TYT ACG
3′-CAC TZT ACG
27.5
27.5
34.0
39.0
35.0
−17.0
−20.0
−16.5
−21.0
−21.5
−15.5
−17.0
−7.5
−12.0
−14.0
−9.0
−16.0
−17.0
−17.0
−14.5
Interestingly, the modified ONs displayed rather different
thermal denaturation characteristics with RNA targets. ONs
modified with 2′-amino-α-L-LNA adenine monomer W
displayed significantly higher affinity against RNA than DNA
targets (ΔTm/modification between +1.5 and +7.0 °C; Table
2). Further, X- and Y-modified ONs displayed markedly lower
thermal affinities toward RNA than DNA targets, with N2′-
PyMe X-modified DNA:RNA duplexes displaying similar
thermostabilities as unmodified reference duplexes (ΔTm/
modification between −0.5 and +2.5 °C; Table 2) and N2′-
a
For conditions of the thermal denaturation experiments, see Table 1.
Tm values for the fully matched duplexes are shown in bold. ΔTm
=
change in Tm relative to the value for the fully matched DNA:DNA
duplex.
X4/Y4/Z4 displayed surprisingly efficient discrimination of
mismatched DNA targets, particularly of AG mismatches
(Table 3), considering the likely intercalative binding mode
of the pyrene moieties, which is known to typically decrease
base-pairing fidelity.9b,26 However, ONs with X/Y/Z mono-
mers positioned as next-nearest neighbors displayed poor
discrimination of the centrally mismatched DNA target (B3
series; Table S4 in the Supporting Information), indicating that
the modification pattern has a major influence on the binding
specificity. Discrimination of RNA mismatches was generally
less efficient with W4/X4/Y4 than with reference ONs but
improved with Z4 (Table 4).
Optical Spectroscopy. UV−vis absorption and steady-
state fluorescence emission spectra of X/Y/Z-modified ONs
and the corresponding duplexes with DNA/RNA targets were
recorded to gain additional insight into the binding modes of
the pyrene moieties of monomers X/Y/Z. Bathochromic shifts
of pyrene absorption maxima, which are indicative of strong
interactions between pyrenes and nucleobases,27 were generally
observed upon hybridization with complementary DNA/RNA
(Δλmax = 0−6 nm; Table 5, Figure 3, and Figures S2 and S3 in
the Supporting Information), with the most pronounced
increases being observed for duplex formation of Y-modified
ONs with DNA. These results are consistent with intercalative
binding modes for the pyrene moieties of monomers X/Y/Z
Table 2. ΔTm Values for Duplexes between B1−B6 and
a
Complementary RNA Targets
ΔTm/°C
ON
duplex
B = W B = X B = Y B = Z
B1
R2
5′-GTG BTA TGC
3′-CAC UAU ACG
+2.5
+2.5
+3.0
+4.5
+7.0
+7.0
−1.0
+1.0
−1.5
−0.5
+2.5
0.0
+1.5
+5.0
+4.5
+2.5
+6.5
+7.0
+4.0
+4.5
B2
R2
5′-GTG ATB TGC
3′-CAC UAU ACG
B3
R2
5′-GTG BTB TGC
3′-CAC UAU ACG
+11.0
+6.5
R1
B4
5′-GUG AUA UGC
3′-CAC TBT ACG
R1
B5
5′-GUG AUA UGC
3′-CAC TAT BCG
+8.0
R1
B6
5′-GUG AUA UGC
3′-CAC TBT BCG
+14.5
a
ΔTm = change in Tm relative to the value for the unmodified
reference duplex D1:R2 (Tm ≡ 26.0 °C) or R1:D2 (Tm ≡ 24.5 °C);
for conditions of the thermal denaturation experiments, see Table 1.
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To further substantiate the proposed intercalative binding
mode of N2′-PyCO monomer Y, we synthesized three
additional Y-modified ONs in which the 3′-flanking nucleotide
was varied systematically (Y7−Y9, Table 6). ONs with 3′-
flanking purines displayed greater relative increases in thermal
affinity against DNA targets than the corresponding ONs with
3′-flanking pyrimidines (compare the ΔTm values for Y7:D3
and Y9:D5 with those for Y8:D4 and Y4:D1 in Table 6). This
is consistent with the proposed binding mode, as 3′-
intercalating pyrenes are expected to interact more strongly
with large purines.
Steady-state fluorescence emission experiments provided
further evidence for an intercalative binding mode of the
pyrenes, as the emission levels of these four DNA duplexes
decreased in the anticipated order of nucleobase quenching
efficiency (from least to most quenched: 3′-flanking A > T > C
> G; Figure 4).28b,29
Table 4. Discrimination of Mismatched RNA Targets by B4
Series and Reference ONs
a
RNA: 5′-GUG ABA UGC
Tm/°C
B = U
ΔTm/°C
B = C
ON
sequence
B = A
B = G
D2
W4
X4
Y4
Z4
3′-CAC TAT ACG
3′-CAC TWT ACG
3′-CAC TXT ACG
3′-CAC TYT ACG
3′-CAC TZT ACG
24.5
29.0
24.0
27.0
31.0
−15.0
−13.5
−10.5
−10.5
−19.0
−15.0
−13.0
−8.0
−11.0
−15.5
−11.0
−11.5
−10.0
−8.5
−14.5
a
For conditions of the thermal denaturation experiments, see Table 1.
Tm values for the fully matched duplexes are shown in bold. ΔTm
=
change in Tm relative to the value for the fully matched RNA:DNA
duplex.
and in agreement with our previous observations for the
corresponding thymine analogues.9b
CONCLUSION
■
Steady-state fluorescence emission spectra were recorded at 5
°C using an excitation wavelength of λex = 350 nm.
Hybridization of N2′-PyMe X-modified or N2′-PyAc Z-
modified ONs with complementary DNA/RNA resulted in
decreased emission (Figure 3 and Figures S4 and S7 in the
Supporting Information), which is consistent with intercalation-
induced quenching by flanking nucleobases.27,28 The resulting
duplexes were only weakly fluorescent and exhibited defined
vibronic bands at ∼380 nm and ∼400 nm along with a shoulder
at 420−425 nm. On the other hand, a range of responses was
observed upon hybridization of N2′-PyCO Y-modified ONs
with complementary DNA/RNA, varying from ∼50% reduc-
tion to ∼3-fold enhancement of emission (Figure S5 in the
Supporting Information). The resulting duplexes were strongly
fluorescent (nearly 2 orders of magnitude more fluorescent
than X-modified duplexes) with little or no vibronic fine
structure (λem,max ≈ 400 nm, broad band). The lack of a
consistent response upon hybridization to DNA/RNA
presumably is not due to different pyrene binding modes in
the resulting duplexes but rather reflects different fluorescence
intensities of the single-stranded probes, possibly due to
different sequence-dependent rotational barriers around the
Py−CO bond. Thus, singly modified DNA duplexes displayed
similar emission intensities (Figure S6 in the Supporting
Information), suggesting that the pyrene moieties are in similar
microenvironments in the duplex, which is consistent with an
intercalative binding mode.
ONs modified with N2′-pyrene-functionalized 2′-amino-α-L-
LNA-adenine monomers X−Z display very high affinity toward
DNA targets. The DNA-selective hybridization, together with
hybridization-induced bathochromic shifts of pyrene absorption
maxima and quenching of pyrene fluorescence, is indicative of
intercalative binding modes for the pyrene moieties. ONs with
such characteristics are likely to be of significant interest for
applications in nucleic acid diagnostics and biotechnology.30
However, the synthesis of the corresponding phosphoramidites
is very challenging because of nontrivial protecting-group
manipulations (<1% overall yield from diacetone-α-D-glucose),
which emphasizes the need for a more efficient synthetic route
toward these building blocks or access to functional analogues
of N2′-functionalized 2′-amino-α-L-LNA monomers, which are
easier to synthesize. In fact, we have already discovered the first
examples of such analogues, namely, N2′-pyrene-functionalized
2′-N-methyl-2′-amino-DNA and O2′-pyrene-functionalized
RNA monomers,8a,12b and are exploring their use for
applications in nucleic acid diagnostics.8b,c
EXPERIMENTAL SECTION
■
9-[2-O-Acetyl-3-O-benzyl-5-O-methanesulfonyl-4-C-metha-
nesulfonyloxymethyl-α-L-threo-pentofuranosyl]-6-N-benzoyla-
denine (6). 6-N-Benzoyladenine (28.6 g, 0.12 mol) and glycosyl
donor 56b (40.6 g, 79.8 mmol) were coevaporated with 1,2-
dichloroethane (2 × 150 mL) and resuspended in anhydrous 1,2-
dichloroethane (270 mL). To this was added N,O-bis(trimethylsilyl)-
Table 5. Absorption Maxima in the 300−400 nm Region for ONs Modified with N2′-Pyrene-Functionalized 2′-Amino-α-L-LNA
a
Adenine Monomers X/Y/Z in the Presence or Absence of Complementary DNA/RNA Targets
λmax/nm [Δλmax/nm]
B = X
+DNA
B = Y
+DNA
B = Z
+DNA
ON
sequence
SSP
+RNA
SSP
+RNA
SSP
+RNA
B1
B2
B3
B4
B5
B6
5′-GTG BTA TGC
5′-GTG ATB TGC
5′-GTG BTB TGC
3′-CAC TBT ACG
3′-CAC TAT BCG
3′-CAC TBT BCG
348
348
347
348
348
348
350 [+2]
350 [+2]
349 [+2]
350 [+2]
350 [+2]
350 [+2]
350 [+2]
349 [+1]
348 [+1]
348 [ 0]
350 [+2]
348 [ 0]
349
349
349
347
350
348
352 [+3]
353 [+4]
351 [+2]
353 [+6]
351 [+1]
352 [+4]
351 [+2]
351 [+2]
351 [+2]
352 [+5]
350 [ 0]
353 [+5]
348
350
347
348
348
348
350 [+2]
351 [+1]
350 [+3]
351 [+3]
351 [+3]
351 [+3]
351 [+3]
351 [+1]
351 [+4]
351 [+3]
351 [+3]
351 [+3]
a
Δλmax = change in absorption maximum relative to λmax for the single-stranded probe (SSP). Measurements were performed at 5 °C except for the
single-stranded X/Y-modified probes, whose spectra were recorded at room temperature. Buffer conditions were the same as in the thermal
denaturation experiments.
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Figure 3. (left) UV−vis absorption and (right) steady-state fluorescence emission spectra of Z4 in the presence or absence of complementary DNA/
RNA. Texp = 5 °C; each ON was used at 1 μM concentration in Tm buffer; λex = 350 nm (fluorescence).
crude residue, which was purified by silica gel column chromatography
Table 6. Tm Values for Duplexes between Y4/Y7/Y8/Y9 and
a
(0−10% v/v i-PrOH in CH2Cl2) to provide nucleoside 6 (38.3 g,
70%) as a white foam. Rf = 0.4 (5% v/v MeOH in CH2Cl2); MALDI-
HRMS m/z 712.1380 ([M + Na]+, C29H31N5O11S2·Na+, calcd
712.1354). The observed 13C NMR data (75.5 MHz, CDCl3) are in
good agreement with previously reported data for this compound.6b
9-[3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfony-
loxymethyl-α-L-threo-pentofuranosyl]-6-N-benzoyladenine (7).
Fully protected nucleoside 6 (4.66 g, 6.76 mmol) was dissolved in a
solution17 of guanidinium nitrate (4.91 g, 40.2 mmol) and NaOMe
(0.24 g, 4.44 mmol) in MeOH:CH2Cl2 (450 mL, 9:1 v/v). The
reaction mixture was stirred at rt for 30 min, at which point sat. aq.
NH4Cl (200 mL) was added. The resulting white precipitate was
filtered off and washed with CH2Cl2, and the filtrate was concentrated.
The aqueous layer was extracted with CH2Cl2 (5 × 200 mL), and the
combined organic layers were evaporated to near dryness. The
resulting crude residue was purified by silica gel column chromatog-
raphy (0−4% v/v MeOH in CH2Cl2) to afford alcohol 7 (3.84 g, 88%)
as a white foam. Rf = 0.5 (10% v/v MeOH in CH2Cl2); MALDI-
HRMS m/z 670.1234 ([M + Na]+, C27H29N5O10S2·Na+, calcd
670.1248). The observed 13C NMR data (75.5 MHz, CDCl3) are in
good agreement with previously reported data for this compound.6b
9-[2-C-Azido-3-O-benzyl-2-deoxy-4-C-methanesulfonyloxy-
methyl-5-O-methanesulfonyl-α-L-erythro-pentofuranosyl]-6-N-
benzoyladenine (8). Alcohol 7 (18.6 g, 28.7 mmol) was
coevaporated with anhydrous pyridine (50 mL) and dissolved in
anhydrous CH2Cl2 (200 mL). The solution was cooled to −78 °C, and
anhydrous pyridine (7.3 mL, 90.6 mmol) was added, followed by
dropwise addition of trifluoromethanesulfonyl anhydride (Tf2O) (9.90
mL, 58.9 mmol). The reaction mixture was allowed to warm to rt and
stirred for 3 h. At this point, crushed ice (50 mL) was added, and the
layers were separated. The organic layer was washed with sat. aq.
NaHCO3 (2 × 50 mL), evaporated to near dryness, and coevaporated
with abs. EtOH (2 × 50 mL) to afford the crude O2′-triflate as a light-
brown residue, which was used in the next step without further
purification.
NaN3 (19.6 g, 0.30 mol) and 15-crown-5 (6.0 mL, 0.30 mol) were
added to a solution of the crude O2′-triflate in anhydrous DMF (300
mL). The reaction mixture was stirred first at rt for 15 h and then for
an additional 8 h at 50 °C. After the mixture was cooled to rt, the
solids were filtered off and washed with EtOAc, and the combined
organic layers were concentrated until near dryness. The concentrate
was taken up in EtOAc (200 mL) and brine (200 mL), and the layers
were separated, after which the aqueous layer extracted with EtOAc (4
× 100 mL). The combined organic layers were evaporated to near
dryness, and the resulting crude residue was purified by silica gel
column chromatography (0−90% v/v EtOAc in petroleum ether) to
afford azide 8 (17.9 g, 89% over two steps) as a white solid material. Rf
= 0.4 (EtOAc); IR (KBr) 2116 cm−1 (N3); MALDI-HRMS m/z
695.1295 ([M + Na]+, C27H28N8O9S2·Na+, calcd 695.1313); 1H NMR
Complementary DNA Targets
ON
duplex
ΔTm/°C
reference Tm/°C
D3
Y7
5′-GTG TT ATGC
3′-CAC AY TACG
+17.0
27.5
D4
Y8
5′-GTG GT ATGC
3′-CAC CY TACG
+10.0
+19.5
+11.5
33.0
D5
Y9
5′-GTG CT ATGC
3′-CAC GY TACG
b
24.0
D1
Y4
5′-GTG AT ATGC
3′-CAC TY TACG
27.5
a
ΔTm = change in Tm relative to the value for the unmodified
b
reference duplex. For experimental conditions, see Table 1. The low
Tm of this reference duplex was confirmed by two independent
operators using strands from three different commercial batches.
Figure 4. Fluorescence emission spectra of duplexes between Y4/Y7/
Y8/Y9 and complementary DNA targets. The nucleotide flanking the
Y monomer on its 3′-side is listed in the parentheses. Texp = 5 °C; each
ON used at 0.15 μM concentration in Tm buffer; λex = 350 nm.
acetamide (BSA) (49.2 mL, 0.20 mol), and the suspension was heated
at reflux until it turned homogeneous. The solution was then cooled to
rt. TMSOTf (43.1 mL, 0.24 mol) was slowly added, and the reaction
mixture was heated at reflux for 70 h. The mixture was then cooled to
rt and slowly poured into a solution of sat. aq. NaHCO3 and crushed
ice (500 mL, 1:1 v/v). Additional crushed ice (∼400 mL) was added,
and the mixture was stirred for 30 min. The resulting precipitate was
removed via filtration, and the filtrate was extracted with CH2Cl2 (1.5
L). The organic layer was washed with sat. aq. NaHCO3 (2 × 500
mL), and the combined aqueous layer was back-extracted with CH2Cl2
(2 × 500 mL). The combined organic layer was evaporated to afford a
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(300 MHz, DMSO-d6) δ 11.30 (s, 1H, ex), 8.79 (s, 1H), 8.54 (s, 1H),
8.05 (d, 2H, J = 7.0 Hz), 7.30−7.67 (m, 8H), 6.74 (d, 1H, J = 4.4 Hz),
5.08−5.16 (m, 1H), 4.89 (d, 1H, J = 5.1 Hz), 4.74−4.83 (m, 2H),
4.69−4.72 (d, 1H, J = 11.4 Hz), 4.47−4.51 (d, 1H, J = 11.4 Hz), 4.42
(s, 2H), 3.28 (s, 3H), 3.24 (s, 3H); 13C NMR (75.5 MHz, DMSO-d6)
δ 151.9, 150.4, 142.7, 136.9, 133.2, 132.5, 128.5, 128.0, 81.9, 81.7, 80.1,
73.5, 68.3, 61.9, 36.98, 36.94. The carbonyl group of the 6-N-benzoyl
group was not visible.
128.34 (Ph), 127.91 (Ph), 127.88 (Ph), 127.53 (Ph), 127.51 (Ph),
125.5, 125.3, 115.5 (q, J = 288 Hz, CF3), 115.2 (q, J = 288 Hz, CF3),
86.16, 86.15, 84.9 (C1′A), 84.0 (C1′B), 79.2 (C3′B), 77.4 (C3′A), 71.6
(CH2Ph), 65.3 (C5′), 65.0 (C5′), 63.0 (C2′B), 61.4 (C2′A), 53.2
(C5″B), 53.1 (C5″A), 37.0 (CH3SO2); 19F NMR (376 MHz, DMSO-
d6) δ −71.3 (CF3,B), −72.1 (CF3,A).
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-hydroxy-1-
methanesulfonyloxymethyl-5-trifluoroacetyl-2-oxa-5-
azabicyclo[2.2.1]heptane (12). Fully protected nucleoside 11 (19.6
g, 30.4 mmol) was coevaporated with 1,2-dichloroethane (3 × 100
mL) and dissolved in anhydrous CH2Cl2 (600 mL). The solution was
cooled to −78 °C, and BCl3 (1 M solution in hexanes, 370 mL, 0.37
mol) was added. The reaction mixture was allowed to warm to rt and
was stirred for 17 h. The mixture was then cooled to 0 °C, and crushed
ice (800 mL) was slowly added. The layers were separated, and the
organic phase was washed with sat. aq. NaHCO3 (2 × 300 mL). The
combined aqueous layers were back-extracted with EtOAc (4 × 500
mL), and the combined organic phases were evaporated to near
dryness. The resulting residue was purified using silica gel column
chromatography (0−20% v/v MeOH in CH2Cl2) to afford alcohol 12
(14.7 g, 87%) as a white solid material. Rf = 0.3 (50% v/v acetone in
CH2Cl2). Physical data for the mixture of rotamers (∼4:6 by 1H
NMR): MALDI-HRMS m/z 579.0871 ([M + Na]+, C21H19F3N6O7S·
Na+, calcd 579.0880); 1H NMR31,32 (500 MHz, DMSO-d6) δ 11.21 (s,
1H, ex, NHA+B), 8.76 (s, 0.6H, H2B), 8.75 (s, 0.4H, H2A), 8.60 (s,
0.4H, H8A), 8.56 (s, 0.6H, H8B), 8.04 (d, 2H, J = 7.7 Hz, BzA+B),
7.48−7.66 (m, 3H, BzA+B), 6.83 (d, 0.6H, J = 1.4 Hz, H1′B), 6.80 (d,
0.4H, J = 1.4 Hz, H1′A), 6.72 (d, 0.6H, ex, J = 4.1 Hz, 3′-OHB), 6.68
(d, 0.4H, ex, J = 4.1 Hz, 3′-OHA), 4.91 (br s, 0.4H, H2′A), 4.82 (d,
0.6H, J = 4.1 Hz, H3′B), 4.76 (d, 0.4H, J = 4.1 Hz, H3′A), 4.70 (br s,
0.6H, H2′B), 4.60−4.67 (m, 2H, H5′A+B), 4.46 (d, 0.4H, J = 10.3 Hz,
H5″A), 4.26 (d, 0.6H, J = 11.7 Hz, H5″B), 4.03 (d, 0.6H, J = 10.3 Hz,
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-benzyloxy-1-
methanesulfonyloxymethyl-2-oxa-5-azabicyclo[2.2.1]heptane
(9). Aqueous NaOH (2 M, 38.9 mL, 77.8 mmol) and trimethylphos-
phine (1 M in THF, 77.8 mL, 77.8 mmol) were added to an ice-cold
solution of azido nucleoside 8 (35.2 g, 51.8 mmol) in THF (500 mL).
The reaction mixture was allowed to warm to rt and was stirred at this
temperature for 21 h. The mixture was then evaporated to near
dryness, and the resulting crude residue taken up in EtOAc (200 mL)
and brine (200 mL). After the layers were separated, the aqueous layer
was extracted with MeOH:CH2Cl2 (3 × 200 mL, 2:8 v/v). The
combined organic layers were evaporated to dryness, and the resulting
crude material was purified by silica gel column chromatography (0−
5% v/v MeOH in CH2Cl2) to provide bicyclic nucleoside 9 (24.2 g,
85%) as a solid brown material. Rf = 0.3 (EtOAc); MALDI-HRMS m/
z 573.1517 ([M + Na]+, C26H26N6O6S·Na+, calcd 573.1527); 1H
NMR31 (500 MHz, DMSO-d6) δ 11.17 (s, 1H, ex, NH), 8.77 (s, 1H,
H8), 8.73 (s, 1H, H2), 8.06 (d, 2H, J = 7.0 Hz, Bz), 7.54−7.67 (m, 3H,
Bz), 7.29−7.47 (m, 5H, Ph), 6.52 (d, 1H, J = 1.8 Hz, H1′), 4.72−4.76
(d, 1H, J = 11.7 Hz, CH2Ph), 4.62−4.67 (d, 1H, J = 11.7 Hz, CH2Ph),
4.57−4.60 (d, 1H, J = 11.7 Hz, H5′A), 4.49−4.53 (d, 1H, J = 11.7 Hz,
H5′B), 4.45 (s, 1H, H3′), 3.93 (br s, 1H, H2′), 3.28−3.31 (m, 1H,
H5″A, partial overlap with H2O), 3.22 (s, 3H, CH3SO2), 3.10−3.13 (d,
1H, J = 9.9 Hz, H5″B); 13C NMR (125 MHz, DMSO-d6) δ 165.5,
152.1, 151.4 (C2), 150.0, 143.1 (C8), 137.8, 133.3, 132.3 (Bz), 128.4
(Ar), 128.2 (Ar), 127.62 (Ar), 127.60 (Ar), 125.1, 87.2, 84.3 (C1′),
80.4 (C3′), 71.0 (CH2Ph), 66.8 (C5′), 59.8 (C2′), 51.1 (C5″), 36.8
H5″A), 3.86 (d, 0.5H, J = 11.7 Hz, H5″B), 3.29 (s, 3H, CH3SO2); 13
C
NMR (125 MHz, DMSO-d6) δ 165.5, 155.3 (q, J = 36.6 Hz, COCF3),
155.1 (q, J = 36.6 Hz, COCF3), 151.7 (C2A/B), 151.6 (C2A/B), 150.3,
150.2, 141.2 (C8B), 141.0 (C8A), 132.4 (Bz), 128.44 (Bz), 128.42
(Bz), 128.39 (Bz), 128.38 (Bz), 125.5, 125.3, 115.5 (q, J = 288 Hz,
CF3), 115.2 (q, J = 288 Hz, CF3), 87.0, 85.8, 84.8 (C1′A), 83.9 (C1′B),
72.5 (C3′B), 70.7 (C3′A), 65.7 (C5′), 65.4 (C5′), 65.3 (C2′B), 63.7
(C2′A), 52.8 (C5″B), 52.6 (C5″A), 37.0 (CH3SO2); 19F NMR (376
MHz, DMSO-d6) δ −71.1 (CF3,B), −72.1 (CF3,A).
1
(CH3SO2). The H and 13C NMR data are in reasonable agreement
with previously reported data from the patent literature.18 The 2-oxo-
5-azabicyclo[2.2.1]heptane skeleton and stereochemistry of 9 were
verified via NOE experiments on downstream product 14.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-benzyloxy-1-
methanesulfonyloxymethyl-5-trifluoroacetyl-2-oxa-5-
azabicyclo[2.2.1]heptane (11). Bicyclic nucleoside 9 (8.68 g, 15.8
mmol) was coevaporated with pyridine (2 × 20 mL) and dissolved in
anhydrous CH2Cl2 (200 mL) and anhydrous pyridine (5.09 mL, 63
mmol). The solution was cooled to 0 °C, and trifluoroacetic acid
anhydride (4.45 mL, 31.5 mmol) was added. The reaction mixture was
stirred for 2 h at 0 °C, at which point crushed ice (50 mL) was added.
The layers were separated, and the organic layer was washed with sat.
aq. NaHCO3 (2 × 50 mL). The combined aqueous layers were back-
extracted with CH2Cl2 (2 × 100 mL) and MeOH:CH2Cl2 (100 mL,
2:8 v/v), and the combined organic layers were evaporated to near
dryness. The resulting crude residue was sequentially coevaporated
with toluene (50 mL) and abs. EtOH:toluene (50 mL, 1:1 v/v) and
purified by silica gel column chromatography (0−100% v/v EtOAc in
petroleum ether) to afford fully protected nucleoside 11 (6.27 g, 62%)
as a white foam. Rf = 0.5 (10% v/v MeOH:EtOAc). Physical data for
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-benzoyloxy-
methyl-7-hydroxy-5-trifluoroacetyl-2-oxa-5-azabicyclo[2.2.1]-
heptane (13). NaOBz (2.99 g, 20.8 mmol) and 15-crown-5 (2.07 mL,
10.4 mmol) were added to a solution of alcohol 12 (5.79 g, 10.4
mmol) in anhydrous DMF (100 mL). The reaction mixture was stirred
first at 90 °C for 5 h and then at rt for an additional 18 h. The mixture
was concentrated to near dryness and taken up in EtOAc and brine.
The layers were separated, and the aqueous layer was extracted with
EtOAc (4 × 200 mL). The combined organic layers were evaporated
to near dryness, and the resulting residue was purified by silica gel
column chromatography (0−3.5% v/v i-PrOH in CHCl3) to afford
O5′-benzoylated nucleoside 13 (5.05 g, 83%) as a white foam. Rf = 0.4
(10% v/v i-PrOH in CHCl3). Physical data for the mixture of rotamers
1
(∼4.5:5.5 by H NMR): MALDI-HRMS m/z 605.1337 ([M + Na]+,
the mixture of rotamers (∼4:6 by H NMR): MALDI-HRMS m/z
C27H21F3N6O6·Na+ calcd 605.1367); 1H NMR31,32 (500 MHz,
DMSO-d6) δ 11.21 (br s, 1H, ex, NHA+B), 8.69 (s, 1H, H2A+B), 8.59
(s, 0.45H, H8A), 8.54 (s, 0.55H, H8B), 8.05−8.13 (m, 4H, BzA+B),
7.49−7.72 (m, 6H, BzA+B), 6.86 (d, 0.55H, J = 1.7 Hz, H1′B), 6.83 (d,
0.45H, J = 1.7 Hz, H1′A), 6.60−6.80 (br s, 1H, ex, 3′-OHA+B), 4.93 (s,
0.55 H, H3′B), 4.89−4.91 (m, 0.9H, H2′A, H3′A), 4.74−4.79 (m, 1.0H,
H5′A+B), 4.68 (br s, 0.55H, H2′B), 4.58−4.64 (m, 1.0H, H5′A+B), 4.54
(d, 0.45H, J = 10.7 Hz, H5″A), 4.35 (d, 0.55H, J = 11.5 Hz, H5″B),
4.10 (d, 0.45H, J = 10.7 Hz, H5″A), 3.93 (d, 0.55H, J = 11.5 Hz,
H5″B); 13C NMR (125 MHz, DMSO-d6) δ 166.5, 165.3, 155.1 (2q, J =
36 Hz, COCF3), 151.8 (C2A/B), 151.6 (C2A/B), 151.4, 140.6 (C8A/B),
140.4 (C8A/B), 134.5, 134.4, 133.6 (Bz), 131.9 (Bz), 129.55 (Bz),
129.54 (Bz), 129.18, 129.17, 128.7 (Bz), 128.4 (Bz), 128.2 (Bz), 125.5,
125.3, 115.5 (2q, CF3, J = 286 MHz, CF3), 87.4, 86.0, 84.5 (C1′A),
83.7 (C1′B), 72.9 (C3′B), 71.0 (C3′A), 65.3 (C2′B), 63.7 (C2′A), 60.7
1
647.1541 ([M + H]+, C28H25F3N6O7S·H+, calcd 647.1530); 1H
NMR31,32 (500 MHz, DMSO-d6) δ 11.21 (s, 0.6H, ex, NHB), 11.19 (s,
0.4H, ex, NHA), 8.78 (s, 0.6H, H2B), 8.76 (s, 0.4H, H2A), 8.63 (s,
0.4H, H8A), 8.60 (s, 0.6H, H8B), 8.05 (d, 2H, J = 7.0 Hz, BzA+B),
7.52−7.67 (m, 3H, BzA+B), 7.31−7.41 (m, 5H, PhA/B), 6.83 (d, 0.6H, J
= 1.1 Hz, H1′B), 6.80 (d, 0.4H, J = 1.1 Hz, H1′A), 5.26 (s, 0.4H, H2′A),
5.17 (s, 0.6H, H2′B), 4.84 (s, 0.6H, H3′B), 4.82 (s, 0.4H, H3′A), 4.62−
4.79 (m, 4H, CH2PhA+B, H5′A+B), 4.53 (d, 0.4H, J = 10.6 Hz, H5″A),
4.35 (d, 0.6H, J = 12.1 Hz, H5″B), 4.07 (d, 0.4H, J = 10.6 Hz, H5″A),
3.91 (d, 0.6H, J = 12.1 Hz, H5″B), 3.28 (s, 3H, CH3SO2); 13C NMR
(125 MHz, DMSO-d6) δ 165.49, 165.48, 155.3 (q, J = 37 Hz,
COCF3), 155.0 (q, J = 37 Hz, COCF3), 151.8 (C2B), 151.65 (C2A),
151.59, 150.4, 150.3, 141.2 (C8B), 141.0 (C8A), 137.2, 137.0, 133.3,
132.4 (Bz), 128.43 (Bz), 128.40 (Ar), 128.39 (Ar), 128.38 (Ar),
12698
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(C5′), 60.2 (C5′), 53.0 (C5″B), 52.9 (C5″A), minor impurities were
observed at δ 121.3, 119.9, and 79.1; 19F NMR (376 MHz, DMSO-d6)
δ −71.1 (CF3,B), −72.0 (CF3,A).
ex, NH), 9.13 (s, 1H, H8), 8.77 (s, 1H, H2), 8.10 (d, 2H, J = 7.5 Hz,
Bz), 7.64−7.67 (t, 1H, J = 7.5 Hz, Bz), 7.52−7.58 (t, 2H, J = 7.5 Hz,
Bz), 6.78−7.41 (m, 22H, DMTr), 6.58 (d, 1H, J = 1.1 Hz, H1′), 6.53
(d, 2H, J = 9.1 Hz, DMTr), 6.43 (d, 2H, J = 9.1 Hz, DMTr), 4.26 (d,
1H, J = 5.0 Hz, H3′), 3.82 (d, 1H, J = 9.9 Hz, H5″), 3.75 (br s, 1H,
H2′), 3.73 (s, 6H, CH3O), 3.65 (s, 3H, CH3O), 3.62 (s, 3H, CH3O),
3.55 (d, 1H, ex, J = 5.0 Hz, 3′-OH), 3.17−3.19 (d, 1H, J = 11.0 Hz,
H5′), 3.09−3.11 (d, 1H, J = 11.0 Hz, H5′), 2.98 (d, 1H, J = 9.9 Hz,
H5″); 13C NMR (125 MHz, DMSO-d6) δ 165.7, 158.0, 157.05,
156.96, 152.0, 151.6 (C2), 150.3, 145.2, 144.8, 142.9 (C8), 136.7,
136.2, 136.0, 135.4, 135.3, 133.3, 132.4 (Bz), 130.5 (DMTr), 130.2
(DMTr), 129.8 (DMTr), 129.7 (DMTr), 128.8 (DMTr), 128.44 (Bz),
128.39 (Bz), 127.73 (DMTr), 127.70 (DMTr), 127.1 (DMTr), 126.5
(DMTr), 126.3, 125.5 (DMTr), 123.8 (DMTr), 113.1 (DMTr), 112.5
(DMTr), 112.4 (DMTr), 88.4, 86.6 (C1′), 85.2, 74.3, 74.0 (C3′), 64.3
(C2′), 60.7 (C5′), 55.0 (C5″), 54.9 (CH3O), 54.8 (CH3O), 54.75
(CH3O), 54.73 (CH3O). A trace impurity of pyridine was identified in
the 13C NMR spectrum at δ 149.5.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-hydroxy-1-hy-
droxymethyl-2-oxa-5-azabicyclo[2.2.1]heptane (14). From 13:
Aqueous NaOH (2 M, 17.5 mL, 0.35 mol) was added to an ice-cold
solution of alcohol 13 (3.41 g, 5.85 mmol) in 1,4-dioxane and water
(225 mL, 2:1 v/v). The reaction mixture was stirred at 0 °C for 2 h, at
which point sat. aq. NH4Cl (25 mL) was added. The solution was
evaporated to dryness, and the resulting crude material was adsorbed
on silica gel and purified by silica gel column chromatography (0−20%
v/v MeOH in CH2Cl2) to afford amino alcohol 14 (1.33 g, 60%) as a
white solid material. Rf = 0.4 (20% v/v MeOH in CH2Cl2); MALDI-
HRMS m/z 405.1294 ([M + Na]+, C18H18N6O4·Na+, calcd 405.1282);
1H NMR (300 MHz, DMSO-d6) δ 11.17 (br s, 1H, ex, NH), 8.73 (s,
1H, ABz), 8.71 (s, 1H, ABz), 8.05 (d, 2H, J = 8.1 Hz, Bz), 7.49−7.67
(m, 3H, Bz), 6.44 (d, 1H, J = 1.9 Hz, H1′), 5.70 (d, 1H, ex, J = 4.0 Hz,
3′-OH), 4.82 (t, 1H, ex, J = 5.5 Hz, 5′-OH), 4.30 (d, 1H, J = 4.0 Hz,
H3′), 3.69 (d, 2H, J = 5.5 Hz, H5′), 3.51 (s, 1H, H2′), 3.15−3.20 (d,
1H, J = 10.6 Hz, H5″), 2.94−3.01 (d, 1H, J = 10.6 Hz, H5″); 13C
NMR (75.5 MHz, DMSO-d6) δ 165.5, 152.0, 151.2, 149.8, 143.2,
133.3, 132.3, 128.4, 125.2, 91.3, 83.9, 73.4, 62.2 (C5″), 58.3 (C2′),
50.6 (C5′).
From 16: Nucleoside 16 (1.00 g, 1.01 mmol) was dissolved in
CHCl2COOH/MeOH/CH3NO2 (50 mL, 3:5:92 v/v/v), and the
solution was stirred at 0 °C for 15 min. Sat. aq. NaHCO3 was carefully
added to neutralize the solution, and the mixture was evaporated to
dryness and adsorbed on silica gel. The resulting residue was purified
by silica gel column chromatography (0−20% v/v MeOH in CH2Cl2,
initially built including 1% v/v Et3N) to afford amino diol 14 as a
white solid material (0.37 g, 96%).
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-(4,4′-dimethoxy-
trityloxymethyl)-7-hydroxy-2-oxa-5-azabicyclo[2.2.1]heptane
(15) and (1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-5-(4,4′-di-
methoxytrityl)-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-
2-oxa-5-azabicyclo[2.2.1]heptane (16). Amino alcohol 14 (1.25 g,
3.27 mmol) was coevaporated with anhydrous pyridine (30 mL) and
redissolved in anhydrous pyridine (65 mL). The solution was cooled
using an ice−salt mixture, and DMTrCl (1.55 g, 4.58 mmol) was
added in one portion (addition over several portions did not influence
reaction outcome). The reaction mixture was warmed to rt and stirred
for 23 h, at which point MeOH (10 mL) was added. The mixture was
diluted with EtOAc (100 mL) and washed with sat. aq. NaHCO3 (2 ×
15 mL). The combined aqueous layers were back-extracted with
EtOAc (3 × 30 mL), and the combined organic phases were
evaporated to dryness and subsequently coevaporated with abs.
EtOH:toluene (3 × 50 mL, 2:1 v/v). The resulting residue was
purified by silica gel column chromatography (0−8% v/v MeOH in
CH2Cl2, initially built with 0.5% v/v Et3N) and subsequently
coevaporated with abs. EtOH:toluene (2 × 50 mL, 1:1 v/v) to afford
nucleoside 15 (0.85 g, 38%) as a white solid material along with
nucleoside 16 (0.97 g, 30%) as a yellow foam.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-(4,4′-dimethoxy-
trityloxymethyl)-5-(9′-fluorenylmethoxycarbonyl)-7-hydroxy-
2-oxa-5-azabicyclo[2.2.1]heptane (17). Amino alcohol 15 (200
mg, 0.29 mmol) was coevaporated in anhydrous pyridine (2 × 2 mL)
and redissolved in anhydrous pyridine (1.5 mL). The solution was
cooled to 0 °C, and 9′-fluorenylmethyl chloroformate (100 mg, 0.38
mmol) added thereto. The reaction mixture was warmed to rt and
stirred for 6 h, at which point it was diluted with EtOAc (30 mL) and
washed with sat. aq. NaHCO3 (30 mL). The aqueous layer was back-
extracted with EtOAc (25 mL), and the combined organic layers were
evaporated to near dryness. The resulting crude material was
coevaporated with abs. EtOH:toluene (2 × 6 mL, 2:1 v/v) and
purified by silica gel column chromatography (30−100% v/v EtOAc in
petroleum ether) to afford target nucleoside 17 (136 mg, 51%) as a
white foam. Rf = 0.4 (EtOAc). Physical data for the mixture of
rotamers (∼1:1.2 by 1H NMR): MALDI-HRMS m/z 929.3241 ([M +
Na]+, C54H46N6O8·Na+, calcd 929.3269); 1H NMR (300 MHz,
DMSO-d6) δ 11.29 (br s, ex), 8.74 (s, 1H), 8.72 (s, 1.2H), 8.55 (s,
1.2H), 8.49 (s, 1H), 6.90−8.08 (m, 57.2H), 6.80 (d, 1.2H, J = 1.7 Hz),
6.73 (d, 1H, J = 1.7 Hz), 6.25 (d, 1.2H, ex, J = 4.4 Hz), 6.22 (d, 1H, ex,
J = 4.4 Hz), 4.50 (br s, 1.2H), 4.45 (br s, 1H), 4.22 (d, 1H, J = 6.9 Hz),
4.15 (d, 1.2H, J = 6.9 Hz), 3.87 (d, 1H, J = 6.9 Hz), 3.83 (d, 1.2H, J =
6.9 Hz), 3.62−3.76 (m, 17.6H), 3.34−3.43 (m, 6.6H); 13C NMR (75.5
MHz, DMSO-d6) δ 165.66, 165.63, 158.1, 154.7, 154.6, 151.8, 151.5,
150.3, 150.2, 144.7, 143.7, 143.6, 143.5, 142.8, 141.3, 141.2, 140.6,
140.4, 140.3, 135.3, 135.2, 133.3, 132.4, 129.7, 128.8, 128.4, 127.9,
127.7, 127.6, 127.5, 127.2, 127.1, 126.9, 126.7, 125.4, 125.2, 125.1,
124.9, 124.6, 121.3, 119.98, 113.2, 88.9, 88.4, 85.4, 84.9, 84.7, 84.6,
72.5, 72.1, 66.8, 66.7, 63.9, 63.4, 60.6, 60.5, 55.0, 52.7, 52.6, 46.4, 45.9.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-(4,4′-dimethoxy-
trityloxymethyl)-7-hydroxy-5-(pyren-1-yl)methyl-2-oxa-5-
azabicyclo[2.2.1]heptane (18). Nucleoside 15 (225 mg, 0.33
mmol) was coevaporated with anhydrous 1,2-dichloroethane (2 × 5
mL) and redissolved in anhydrous 1,2-dichloroethane (4 mL). 1-
Pyrenecarboxaldehyde (115 mg, 0.49 mmol) and NaBH(OAc)3 (104
mg, 0.49 mmol) were added, and the resulting suspension was stirred
at rt for 17 h, at which point sat. aq. NaHCO3 (30 mL) was added.
The mixture was extracted with CH2Cl2 (3 × 15 mL), and the
combined organic layers were dried (Na2SO4) and evaporated to
dryness. The resulting residue was purified by silica gel column
chromatography (0−5% v/v MeOH in CH2Cl2) to afford N2′-
alkylated nucleoside 18 as a white foam (201 mg, 68%). Rf = 0.4
(EtOAc); MALDI-HRMS m/z 921.3326 ([M + Na]+, C56H46N6O6·
Physical data for 15: Rf = 0.4 (10% v/v MeOH in CH2Cl2);
MALDI-HRMS m/z 707.2609 ([M + Na]+, C39H36N6O6·Na+, calcd
31
1
707.2589); H NMR (500 MHz, DMSO-d6) δ 11.16 (br s, 1H, ex,
NH), 8.78 (s, 1H, H8), 8.73 (s, 1H, H2), 8.06 (d, 2H, J = 7 Hz, Bz),
7.63−7.67 (t, 1H, J = 7.7 Hz, Bz), 7.54−7.58 (d, 2H, J = 7.0 Hz, Bz),
7.19−7.44 (m, 9H, DMTr), 6.86−6.92 (m, 4H, DMTr), 6.54 (d, 1H, J
= 1.9 Hz, H1′), 5.66 (d, 1H, ex, J = 4.8 Hz, 3′-OH), 4.40 (d, 1H, J =
4.8 Hz, H3′), 3.74 (s, 6H, CH3O), 3.50 (br s, 1H, H2′), 3.30−3.32 (m,
1H, H5′, overlap with H2O), 3.25−3.27 (d, 1H, J = 10.7 Hz, H5′),
3.19 (br s, 2H, H5″), 2.90 (br s, 1H, ex, 2′-NH); 13C NMR (125 MHz,
DMSO-d6) δ 165.5, 158.0, 152.2, 151.3 (C2), 149.9, 144.8, 143.3
(C8), 135.5, 135.4, 133.4, 132.3 (Ar), 129.71 (Ar), 129.68 (Ar),
128.45, 128.39 (Ar), 127.8 (Ar), 127.7 (Ar), 126.6 (Ar), 125.2, 113.2
(DMTr), 89.6, 85.1, 84.1 (C1′), 74.0 (C3′), 62.2 (C2′), 61.2 (C5′),
55.0 (CH3O), 51.2 (C5″).
31
1
Na+, calcd 921.3371); H NMR (500 MHz, DMSO-d6) δ 11.18 (s,
1H, ex, NH), 8.53 (s, 1H, H2), 8.46 (s, 1H, H8), 7.97−8.24 (m, 8H,
Ar), 7.60−7.85 (m, 6H, Ar), 7.41−7.44 (m, 2H, DMTr), 7.19−7.32
(m, 7H, DMTr), 6.85−6.92 (m, 4H, DMTr), 6.50 (d, 1H, J = 1.9 Hz,
H1′), 6.14 (d, 1H, ex, J = 3.6 Hz, 3′-OH), 4.72−4.75 (d, 1H, J = 12.5
Hz, CH2Py), 4.60 (d, 1H, J = 3.6 Hz, H3′), 4.52−4.56 (d, 1H, J = 12.5
Hz, CH2Py), 3.73 (s, 6H, CH3O), 3.66 (s, 1H, H2′), 3.34−3.45 (m,
3H, 2 × H5′, H5″), 3.17−3.20 (d, 1H, J = 10.3 Hz, H5″); 13C NMR
(125 MHz, DMSO-d6) δ 165.6, 158.0, 151.7, 151.0 (C2), 149.9, 144.8,
Physical data for 16: Rf = 0.5 (3% v/v MeOH in CH2Cl2); MALDI-
HRMS m/z 1009.3900 ([M + Na]+, C60H54N6O8·Na+, calcd
31
1
1009.3895); H NMR (500 MHz, DMSO-d6) δ 11.33 (br s, 1H,
12699
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142.8 (C8), 135.5, 135.4, 133.6, 133.2, 132.4 (Ar), 130.6, 130.2, 130.0,
129.71 (DMTr), 129.68 (DMTr), 128.7, 128.5 (Ar), 128.4 (Ar), 127.8
(DMTr), 127.7 (DMTr), 127.6 (Ar), 127.2 (Ar), 126.9 (Ar), 126.8
(Ar), 126.6 (DMTr), 125.9 (Ar), 125.5, 124.9 (Ar), 124.8 (Ar), 124.2
(Ar), 123.9, 123.7, 123.1 (Ar), 113.2 (DMTr), 90.3, 85.2, 84.8 (C1′),
75.4 (C3′), 66.3 (C2′), 61.3 (C5′), 59.2 (C5″), 58.2 (CH2Py), 55.0
(CH3O).
127.85 (Ar), 127.77 (Ar), 127.72 (Ar), 127.4 (Ar), 127.27 (Ar), 127.2
(Ar), 127.1 (Ar), 126.9, 126.8 (Ar), 126.68 (Ar), 126.2, 126.0 (Ar),
125.9 (Ar), 125.4, 125.3, 125.00 (Ar), 124.98 (Ar), 124.82 (Ar),
124.76 (Ar), 124.7 (Ar), 124.6 (Ar), 124.5 (Ar), 124.4 (Ar), 124.3
(Ar), 124.1, 123.89, 123.79, 123.76, 123.72, 123.2 (Ar), 113.2 (Ar),
89.0, 88.4, 85.5, 85.4, 84.73 (C1′A), 84.66 (C1′B), 72.9 (C3′A), 72.0
(C3′B), 64.5 (C2′A), 61.8 (C2′B), 60.9 (C5′B), 60.8 (C5′A), 55.0
(CH3O), 52.8 (C5″B), 52.3 (C5″A), 37.8 (CH2PyB), 37.7 (CH2PyA).
Trace impurities of dichloromethane and 1-pyreneacetic acid were
identified in the 13C NMR spectrum. The compound was used in the
next step without further purification.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-[2-cyanoethoxy-
(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxy-
methyl)-5-(9″-fluorenylmethoxycarbonyl)-2-oxa-5-azabicyclo-
[2.2.1]heptane (1). Nucleoside 17 (225 mg, 0.25 mmol) was
coevaporated with anhydrous 1,2-dichloroethane (2 × 4 mL) and
redissolved in anhydrous CH2Cl2 (3.5 mL). Anhydrous N,N′-
diisopropylethylamine (DIPEA) (195 μL, 1.12 mmol) and N-
methylimidazole (NMI) (16 μL, 0.20 mmol) were added, followed
by dropwise addition of 2-cyanoethyl-N,N′-diisopropylchlorophos-
phoramidite (111 μL, 0.50 mmol). The reaction mixture was stirred
for 4 h at rt and evaporated to dryness, and the resulting residue was
purified by silica gel column chromatography (0−2% v/v MeOH in
CH2Cl2 v/v, initially built in 0.5% v/v Et3N) and subsequent
precipitation from CH2Cl2/petroleum ether to afford target amidite
1 (126 mg, 46%) as a white foam. Physical data for mixture of
rotamers: Rf = 0.5 (5% v/v MeOH in CH2Cl2); ESI-HRMS m/z
1129.4356 ([M + Na]+, C63H63N8O9P·Na+, calcd 1129.4348); 31P
NMR (121 MHz, CDCl3) δ 150.32, 150.26, 150.1, 149.5.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-[2-cyanoethoxy-
(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxy-
methyl)-5-(pyren-1-yl)methyl-2-oxa-5-azabicyclo[2.2.1]-
heptane (2). Nucleoside 18 (155 mg, 0.17 mmol) was coevaporated
with 1,2-dichloroethane (2 × 5 mL) and dissolved in a 20% v/v N,N′-
diisopropylethylamine solution in anhydrous CH2Cl2 (5.0 mL). To
this was added 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite
(0.10 mL, 0.44 mmol), and the reaction mixture was stirred at rt for 17
h, whereupon the reaction was quenched with abs EtOH (1 mL) and
the mixture was evaporated to dryness. The resulting residue was
purified by silica gel column chromatography (0−90% v/v EtOAc in
petroleum ether) and precipitated from CH2Cl2/petroleum ether to
afford amidite 2 as a white solid material (131 mg, 69%). Rf = 0.7 (5%
v/v MeOH in CH2Cl2); ESI-HRMS m/z 1099.4642 ([M + H]+,
C65H63N8O7P·H+, calcd 1099.4630); 31P NMR (121 MHz, CDCl3) δ
150.6, 148.4.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-[2-cyanoethoxy-
(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxy-
methyl)-5-(pyren-1-yl)carbonyl-2-oxa-5-azabicyclo[2.2.1]-
heptane (3). Nucleoside 19 (0.30 g, 0.33 mmol) was coevaporated
with 1,2-dichloroethane (2 × 5 mL) and dissolved in 20% v/v N,N′-
diisopropylethylamine in anhydrous CH2Cl2 (3.4 mL). To this was
added 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite (0.11
mL, 0.49 mmol), and the reaction mixture was stirred for 22 h at rt,
at which point abs. EtOH (2 mL) was added. The mixture was taken
up in CH2Cl2 (50 mL) and washed with sat. aq. NaHCO3 (50 mL)
and brine (50 mL). The combined aqueous layers were back-extracted
with CH2Cl2 (2 × 20 mL), and the combined organic layers were
evaporated to dryness. The resulting residue was purified by silica gel
column chromatography (0−99% v/v EtOAc in petroleum ether
containing 1% v/v pyridine), coevaporated with abs. EtOH:toluene (2
× 50 mL, 1:1 v/v), and precipitated from EtOAc/hexane to afford
phosphoramidite 3 as a white foam (247 mg, 67%). Physical data for
the mixture of rotamers: Rf = 0.5 (EtOAc); ESI-HRMS m/z 1113.4462
([M + H]+, C65H61N8O8P·H+, calcd 1113.4422); 31P NMR (121 MHz,
CDCl3) δ 152.3, 151.8, 150.2.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-(4,4′-dimethoxy-
trityloxymethyl)-7-hydroxy-5-(pyren-1-yl)carbonyl-2-oxa-5-
azabicyclo[2.2.1]heptane (19). Amino alcohol 15 (0.41 g, 0.59
mmol) was coevaporated with anhydrous 1,2-dichloroethane (2 × 10
mL) and dissolved in anhydrous CH2Cl2 (11.9 mL), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (226
mg, 1.19 mmol) and 1-pyrenecarboxylic acid (0.29 g, 1.19 mmol) were
added thereto. The reaction mixture was stirred for 45 h at rt, at which
point it was diluted with CH2Cl2 (50 mL) and washed with water (20
mL). The two phases were separated, and the aqueous phase was back-
extracted with CH2Cl2 (3 × 50 mL). The combined organic phases
were evaporated to dryness, and the resulting residue was purified by
silica gel column chromatography (0−99% v/v EtOAc and 1% v/v
pyridine in petroleum ether). The resulting product was coevaporated
with abs. EtOH:toluene (2 × 50 mL, 1:1 v/v) to afford nucleoside 19
(343 mg, 64%) as a yellow solid material. Physical data for the mixture
of rotamers (∼0.4:1 by 1H NMR): Rf = 0.5 (5% v/v MeOH:CH2Cl2);
MALDI-HRMS m/z 935.3138 ([M + Na]+, C56H44N6O7·Na+, calcd
31,32
1
935.3164). H NMR
(300 MHz, DMSO-d6) δ 11.21 (br s, 1.4H,
ex, NH), 8.55 (s, 1H, ABzB), 8.53 (s, 0.4H, ABzA), 8.45 (s, 1H, ABzB),
8.40 (s, 0.4H, ABzA), 6.82−8.23 (m, ∼37.8H, ArA+B), 6.50 (d, 1H, J =
2.2 Hz, H1′B), 6.40 (d, 0.4H, J = 1.8 Hz, H1′A), 6.15 (d, 1H, ex, J = 4.0
Hz, 3′-OHB), 6.07 (d, 0.4H, ex, J = 3.7 Hz, 3′-OHA), 4.42−4.77 (m,
4.2H, H3′A+B, H5′A+B), 3.55−3.75 (m, ∼9.8H, CH3OA+B, H2′A+B),
3.15−3.44 (m, ∼2.8H, H5″A+B); 13C NMR (75.5 MHz, DMSO-d6) δ
165.5, 157.9, 157.7, 151.7, 150.98, 149.8, 148.2, 144.7, 142.8, 140.1,
135.4, 135.3, 133.5, 133.1, 132.3, 130.5, 130.2, 129.9, 129.6, 128.8,
128.6, 128.4, 127.8, 127.6, 127.5, 127.3, 127.2, 126.9, 126.5, 126.3,
125.9, 125.5, 124.9, 124.8, 124.78, 124.2, 123.8, 123.7, 123.1, 113.1,
112.7, 92.0, 90.3, 85.1, 84.7, 79.8, 75.3, 74.7, 66.4, 61.2, 59.1, 58.6,
58.3, 58.2, 54.6.
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-1-(4,4′-dimethoxy-
trityloxymethyl)-7-hydroxy-5-(pyren-1-yl)acetyl-2-oxa-5-
azabicyclo[2.2.1]heptane (20). Amino alcohol 15 (0.25 g, 0.37
mmol) was coevaporated with anhydrous 1,2-dichloroethane (2 × 5
mL) and dissolved in anhydrous CH2Cl2 (10 mL). To this was added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·
HCl) (108 mg, 0.55 mmol) and 1-pyreneacetic acid (145 mg, 0.55
mmol). The reaction mixture was stirred for 2.5 h at rt, at which point
it was diluted with CH2Cl2 (20 mL) and washed with water (2 × 10
mL). The aqueous layer was back-extracted with CH2Cl2 (10 mL), and
the combined organic layers were evaporated to dryness. The resulting
crude residue was purified by silica gel column chromatography (0−
4% v/v MeOH in CH2Cl2) to afford nucleoside 20 as a white solid
(0.27 g, 79%). Physical data for the mixture of rotamers (∼0.5:1.0 by
1H NMR): Rf = 0.5 (10% v/v MeOH:CH2Cl2); MALDI-HRMS m/z
949.3321 ([M + Na]+, C57H46N6O7·Na+, calcd 949.3320); 1H
NMR31,32 (500 MHz, DMSO-d6) δ 11.29 (br s, 1.5H, ex, NHA+B),
8.97 (s, 1H, H8B), 8.73 (s, 0.5H, H2A), 8.71 (s, 1H, H2B), 8.55 (s,
0.5H, H8A), 6.91−8.32 (m, 40.5H, ArA+B), 6.83 (d, 0.5H, J = 1.7 Hz,
H1′A), 6.80 (d, 1H, J = 1.4 Hz, H1′B), 6.32 (d, 0.5H, ex, J = 4.4 Hz, 3′-
OHA), 6.19 (d, 1H, ex, J = 4.4 Hz, 3′-OHB), 5.07 (s, 0.5H, H2′A),
4.71−4.73 (m, 1.5H, H2′B, H3′A), 4.67 (d, 1H, J = 10.4 Hz, H5″B),
4.63 (d, 1H, J = 4.4 Hz, H3′B), 4.55−4.60 (d, 1H, J = 17.0 Hz,
CH2PyB), 4.36−4.40 (d, 1H, J = 17.0 Hz, CH2PyB), 4.11 (d, 0.5H, J =
16.2 Hz, CH2PyA), 3.96−4.03 (m, 1.5H, H5″A, H5″B), 3.66−3.77 (m,
9.5H, CH3O, H5″A), 3.34−3.48 (m, 3.5H, H5′A+B, CH2PyA); 13C
NMR (125 MHz, DMSO-d6) δ 169.9, 169.6, 165.6, 165.5, 158.1,
158.0, 152.0, 151.7 (ABzA), 151.5, 151.4 (ABzB), 150.6, 150.2, 144.71,
144.68, 141.7 (ABzB), 141.5 (ABzA), 135.4, 135.2, 133.54, 133.47, 132.5
(Ar), 130.70, 130.69, 130.28, 130.1, 129.91, 129.88, 129.82 (Ar),
129.79 (Ar), 129.77, 129.74 (Ar), 129.68, 129.62, 129.4, 129.2, 128.8,
128.7, 128.58 (Ar), 128.56 (Ar), 128.54 (Ar), 128.46 (Ar), 128.2 (Ar),
(1S,3R,4S,7R)-3-(6-N-Benzoyladenin-9-yl)-7-[2-cyanoethoxy-
(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytrityloxy-
methyl)-5-(pyren-1-yl)acetyl-2-oxa-5-azabicyclo[2.2.1]heptane
(4). Nucleoside 20 (235 mg, 0.25 mmol) was coevaporated with
anhydrous 1,2-dichloroethane (2 × 5 mL) and redissolved in
anhydrous CH2Cl2 (5 mL). Anhydrous N,N′-diisopropylethylamine
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(220 μL, 1.27 mmol) was added, followed by dropwise addition of 2-
cyanoethyl-N,N′-diisopropylchlorophosphoramidite (115 μL, 0.51
mmol). The reaction mixture was stirred at rt for 22 h, at which
point abs. EtOH (1 mL) was added. The solution was evaporated to
dryness, and the resulting crude residue was purified by silica gel
column chromatography (0−2% v/v MeOH in CH2Cl2) and
precipitation from CH2Cl2/petroleum ether to afford target amidite
4 (203 mg, 71%) as a white foam. Physical data for the mixture of
rotamers: Rf = 0.6 (5% v/v MeOH in CH2Cl2); MALDI-HRMS m/z
1149.4358 ([M + Na]+, C66H63N8O8P·Na+, calcd 1149.4399); 31P
NMR (121 MHz, CDCl3) δ 150.4, 150.31, 150.26, 148.1.
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hrdlicka@uidaho.edu.
■
Author Contributions
§N.K.A. and B.A.A contributed equally.
Notes
The authors declare no competing financial interest.
Synthesis of ONs. W/X/Y/Z-modified oligodeoxyribonucleotides
were made on an automated DNA synthesizer using 0.2 μmol scale
succinyl-linked long-chain alkylamine controlled pore glass (LCAA-
CPG) columns with a pore size of 500 Å. Phosphoramidites 1−4 were
incorporated into ONs using the following hand-coupling conditions
(activator; coupling time; stepwise coupling yield): monomer W
(pyridinium hydrochloride; 30 min; ∼82%); monomers X−Z
(pyridinium hydrochloride; 15 min; ∼95%). Standard protocols for
incorporation of DNA phosphoramidites were used. Modified ONs
were deprotected using 32% aq. NH3 (55 °C, 2−12 h) and purified
(DMTr-ON) by ion-pair reversed-phase HPLC using either an
ammonium formate/acetonitrile gradient or a triethylammonium
acetate/acetonitrile gradient, which was followed by detritylation
(80% aq. AcOH, 20 min) and precipitation (abs. EtOH or acetone,
−18 °C, 12 h). The compositions of the modified ONs were verified
by MALDI-MS analysis (Tables S1 and S2 in the Supporting
Information) recorded in either positive or negative ion mode on a
quadrupole time-of-flight tandem mass spectrometer equipped with a
MALDI source using 3-hydroxypicolinic acid as a matrix. Purity (>90%
unless stated otherwise) was verified either by the ion-pair reversed-
phase HPLC system running in analytical mode or ion-exchange
HPLC using a Tris-Cl/EDTA−NaCl gradient.
Protocol for Thermal Denaturation Studies. Concentrations of
ONs were calculated using the following extinction coefficients
(OD260/μmol): G, 12.0; A, 15.2; T, 8.4; U, 10.0; C, 7.1; pyrene,
22.4. ONs (each strand at 1.0 μM) were thoroughly mixed, denatured
by heating, and subsequently cooled to the starting temperature of the
experiment. Quartz optical cells with a path length of 10.0 mm were
used. The thermal denaturation temperature (Tm/°C) was measured
on a temperature-controlled UV−vis spectrophotometer and
determined as the maximum of the first derivative of the thermal
denaturation curve (A260 vs T) recorded in medium-salt buffer (Tm
buffer) (100 mM NaCl, 0.1 mM EDTA, and pH 7.0 adjusted with 10
mM NaH2PO4/5 mM Na2HPO4). The temperature of the
denaturation experiments ranged from at least 15 °C below Tm to
20 °C above Tm (although not below 3 °C). A temperature ramp of
0.5 °C/min or 1.0 °C/min was used in the experiments. The reported
thermal denaturation temperatures are averages of two measurements
within 1.0 °C.
ACKNOWLEDGMENTS
■
P.J.H. appreciates financial support through Award R01
GM088697 from the National Institute of General Medical
Sciences, National Institutes of Health, and from the Institute
of Translational Health Sciences (ITHS) (supported by Grants
UL1 RR025014, KL2 RR025015, and TL1 RR025016 from the
National Center for Research Resources, NIH). J.W.
appreciates financial support from The Danish National
Research Foundation and the Danish Agency for Science
Technology and Innovation. We thank Dr. T. Santhosh Kumar
(University of Southern Denmark) for providing us with
starting material and Dr. Lee Deobald (EBI Murdock Mass
Spectrometry Center, University of Idaho) for mass spectro-
metric analyses.
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ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental section; additional synthetic strategies;
NMR spectra for all new compounds; MS data for all modified
ONs; representative thermal denaturation curves; additional Tm
data; representative absorption and fluorescence emission
spectra; and evaluation of SNP-discriminatory potential. This
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