High-affinity DNA targeting using readily accessible mimics of N2′-functionalized 2′-amino-α-L-LNA

Article abstract of DOI:10.1021/jo201095p

N2′-Pyrene-functionalized 2′-amino-α-L-LNAs (locked nucleic acids) display extraordinary affinity toward complementary DNA targets due to favorable preorganization of the pyrene moieties for hybridization- induced intercalation. Unfortunately, the synthes

Full text of DOI:10.1021/jo201095p

ARTICLE
pubs.acs.org/joc
High-Affinity DNA Targeting Using Readily Accessible Mimics of
N20-Functionalized 20-Amino-r-L-LNA
Saswata Karmakar,† Brooke A. Anderson,†,§ Rie L. Rathje,†,‡,§ Sanne Andersen,†,‡,§ Troels B. Jensen,‡
Poul Nielsen,‡ and Patrick J. Hrdlicka*,†
†Department of Chemistry, University of Idaho, Moscow, Idaho 83844, United States
‡Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark
S Supporting Information
b
ABSTRACT: N20-Pyrene-functionalized 20-amino-R-L-LNAs (locked
nucleic acids) display extraordinary affinity toward complementary
DNA targets due to favorable preorganization of the pyrene moieties
for hybridization-induced intercalation. Unfortunately, the synthesis of
these monomers is challenging (∼20 steps, <3% overall yield), which has
precluded full characterization of DNA-targeting applications based on
these materials. Access to more readily accessible functional mimics would
be highly desirable. Here we describe short synthetic routes to a series of
O20-intercalator-functionalized uridine and N20-intercalator-functional-
ized 20-N-methyl-20-aminouridine monomers and demonstrate, via ther-
mal denaturation, UVꢀvis absorption and fluorescence spectroscopy experiments, that several of them mimic the DNA-
hybridization properties of N20-pyrene-functionalized 20-amino-R-L-LNAs. For example, oligodeoxyribonucleotides (ONs)
modified with 20-O-(coronen-1-yl)methyluridine monomer Z, 20-O-(pyren-1-yl)methyluridine monomer Y, or 20-N-(pyren-1-
ylmethyl)-20-N-methylaminouridine monomer Q display prominent increases in thermal affinity toward complementary DNA
relative to reference strands (average ΔTm/mod up to +12 °C), pronounced DNA-selectivity, and higher target specificity than 20-
amino-R-L-LNA benchmark probes. In contrast, ONs modified with 20-O-(2-napthyl)uridine monomer W, 20-O-(pyren-1-
yl)uridine monomer X or 20-N-(pyren-1-ylcarbonyl)-20-N-methylaminouridine monomer S display very low affinity toward
DNA targets. This demonstrates that even conservative alterations in linker chemistry, linker length, and surface area of the
appended intercalators have marked impact on DNA-hybridization characteristics. Straightforward access to high-affinity building
blocks such as Q, Y, and Z is likely to accelerate their use in DNA-targeting applications within nucleic acid based diagnostics,
therapeutics, and material science.
’ INTRODUCTION
LNA” approach.12 The synthesis of the N20-intercalator-func-
tionalized 20-amino-R-L-LNA thymine phosphoramidites is un-
fortunately very challenging. For example, the corresponding
phosphoramidite of 20-N-(pyren-1-yl)methyl-20-amino-R-L-LNA
thymine monomer L is obtained in ∼3% yield over ∼20 steps
from diacetone-R-D-glucose (Figure 1).9b,13 Access to syntheti-
cally more readily accessible mimics of N20-intercalator-
functionalized 20-amino-R-L-LNA monomers would be highly
desirable to evaluate the full scope of the aforementioned DNA-
targeting applications.
Development of chemically modified oligonucleotides con-
tinues to attract remarkable attention due to their wide-ranging
applications within fundamental research, diagnostics, therapeu-
tics, and materials science.1 Representative applications include
modulation of gene expression at the DNA2 or RNA level,3
detection of DNA/RNA targets including those with single
nucleotide polymorphisms (SNPs),4 and the use of nucleic acids
as supramolecular scaffolds for organization of chromophores.5
As part of our ongoing interest in functionalized variants6 of
affinity- and specificity-enhancing LNA (locked nucleic acid)7
and R-L-LNA,7c,8 we recently developed oligodeoxyribonucleo-
tides (ONs) modified with N20-pyrene-functionalized 20-amino-
R-L-LNA monomers.9 The ability of these monomers to pre-
cisely and site-specifically position intercalators in DNA duplex
cores results in exceptional affinity toward complementary
DNA (cDNA) targets and has enabled us to (a) form dense
pyrene arrays within duplex cores,10 (b) develop probes for
fluorescent discrimination of SNPs in DNA targets under
nonstringent conditions,11 and (c) target mixed-sequence
regions in double-stranded DNA via the so-called “Invader
We identified O20-intercalator-functionalized RNA and N20-
intercalator-functionalized 20-N-methyl-20-amino-DNA mono-
mers (Figure 1) as potential structural and functional mimics
of N20-intercalator-functionalized 20-amino-R-L-LNA. We based
this hypothesis on studies showing (a) that ONs modified with
20-O-(pyren-1-yl)methyluridine monomer Y or 20-N-methyl-20-
N-(pyren-1-ylmethyl)-20-amino-DNA monomer Q display high
thermal affinity toward cDNA,14,15 and (b) that the pyrene
Received: May 28, 2011
Published: August 09, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of monomers studied herein: 20-N-(pyren-1-yl)methyl-20-amino-R-L-LNA thymine monomer L, O20-intercalator-
functionalized uridine monomers WꢀZ, and N20-intercalator-functionalized 20-N-methyl-20-aminouridine monomers Q, S, and V.
Scheme 1. Synthesis of O20-Intercalator-Functionalized Uridine Phosphoramidites 4Wꢀ4Z
moieties intercalate between the nucleobase of the monomer and
the 30-flanking nucleoside.16 Motivated by this, we set out to (a)
develop synthetic routes to a series of O20-intercalator-function-
alized uridine and N20-intercalator-functionalized 20-N-methyl-20-
aminouridine phosphoramidites and (b) compare hybridization
properties of the correspondingly modified ONs with those of
20-N-(pyren-1-yl)methyl-20-amino-R-L-LNA “benchmark” probes
(Figure 1).
Yamana and co-workers previously installed arylmethyl
groups at the O20-position of 30,50-ditrityluridine via Williamson
etherification.17,21 We found the initial O30,O50-tritylation of
uridine22 and the protecting group manipulations following the
O20-functionalization step to be cumbersome. Instead, the reac-
tion between 1 and tris(pyrene-1-ylmethyl) or tris(coronene-1-
ylmethyl) borate, generated in situ upon addition of pyren-1-
ylmethanol23 or coronen-1-ylmethanol24 to borane, afforded 2Y
and 2Z in modest but robust yields (Scheme 1).25 The two-step
route to nucleoside 2Y from uridine proceeds in similar yield
(∼20%) as the original three-step approach17 but is more con-
venient. To our knowledge, this is the first time that the trialkyl
borate mediated opening26 of O2,O20-anhydrouridine 1 has
been used to install large arylmethylethers at the C20-position
of nucleosides. Subsequent O50-dimethoxytritylation afforded
nucleosides 3Wꢀ3Z in 47ꢀ78% yield, which upon treatment
with 2-cyanoethyl-N,N-diisopropylchlorophosporamidite (PCl
reagent) and H€unig’s base provided target phosphoramidites
4Wꢀ4Z in 74ꢀ78% yield (Scheme 1).
’ RESULTS AND DISCUSSION
Synthesis of O20-Intercalator-Functionalized Uridine
Phosphoramidites. The four phosphoramidites 4Wꢀ4Z were
selected as synthetic targets in this series as this allowed us to
(a) test whether known monomer Y17 indeed is a mimic of
“benchmark” monomer L and (b) systematically evaluate the
influence of linker length (monomer X vs Y) and aromatic
surface area (monomer W vs X; monomer Y vs Z) on the
hybridization properties of modified ONs (Scheme 1).
Sekine and co-workers recently described a microwave-based
approach in which 2,20-anhydrouridine 1 is treated with neat
phenols to afford O20-arylated uridines.18 We adapted this
approach for conventional heating sources and used 2-naphtol
and 1-pyrenol19 to obtain 2W18 and 2X in 25% and 44% yield,
respectively (Scheme 1). Treatment of 120 with pyren-1-yl-
methanol or coronen-1-ylmethanol under similar conditions
failed to afford desired products 2Y and 2Z in acceptable yields,
requiring development of an alternative approach.
Synthesis of N20-Pyrene-Functionalized 20-N-Methyl-20-
aminodeoxyuridine Phosphoramidites. In this series, three
phosphoramidites 7Q, 7S, and 7V were selected as synthetic
targets to (a) evaluate whether known monomer Q15 is a mimic
of benchmark monomer L and (b) to study the influence of link-
er chemistry (monomer Q vs S) and length (monomer S vs V)
on hybridization properties of correspondingly modified ONs
(Scheme 2).
Wengel and co-workers previously reported a route to 20-N-
methyl-20-N-(pyren-1-ylmethyl)-20-aminouridine phosphoramidite
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Scheme 2. Synthesis of N20-Intercalator-Functionalized Uridine Phosphoramidites 7Q, 7S, and 7V
7Q from 50-O-dimethoxytrityl-20-aminouridine, which entails
N20-functionalization via reductive amination, O50-detritylation,
N20-methylation under acidic conditions, O50-tritylation, and
O30-phophitylation (∼7% overall yield over five steps).15 In order
to decrease the number of protection/deprotection steps and to
introduce N20-moieties at a later stage, we instead decided to
utilize nucleoside 5 as a starting material, which is easily obtained
from 50-O-dimethoxytrityl-2,20-anhydrouridine in ∼73% yield
over three steps.27 Direct N20-alkylation of 5 using pyren-1-
ylmethyl chloride afforded the desired product 6Q in 46% yield.25
Interestingly, reductive amination of 5 using 1-pyrenecarbalde-
hyde and sodium triacetoxyborohydride28 or sodium cyanobor-
ohydride failed to afford 6Q in acceptable yields due to prominent
formation of the corresponding cyclic N20,O30-hemiaminal ether
(see Supporting Information, Scheme S1). Although formation
of this byproduct was avoided by prior protection of the
O30-position of 5 as a TBDMS-ether, the increased steric bulk
resulted in low yields during the subsequent reductive amination
(results not shown).
HATU-mediated coupling between nucleoside 5 and 1-pyr-
enecarboxylic acid afforded N20-acylated nucleoside 6S in 78%
yield, while EDC-mediated coupling between nucleoside 5
and 1-pyreneacetic acid furnished 6V in 83% yield.25 Subse-
quent O30-phosphitylation of 6Q, 6S, and 6V using similar
conditions as for the synthesis of 4Wꢀ4Z afforded phosphor-
amidites 7Q, 7S, and 7V although only in moderate yields
(42ꢀ57%), presumably due to the increased steric bulk at the
N20-position.
Synthesis of Modified ONs and Setup of Thermal Dena-
turation Studies. Intercalator-functionalized phosphoramidites
4W, 4X, 4Y, 4Z, 7Q, 7S, and 7V were incorporated into ONs via
machine-assisted solid-phase DNA synthesis using the following
hand-coupling conditions (activator; coupling time; approximate
coupling yield): monomer W/X/Y (4,5-dicyanoimidazole; 15 min;
∼98%/∼98%/∼98%); monomer Z(4,5-dicyanoimidazole; 35 min;
∼90%); monomers Q, S, and V (5-(bis-3,5-trifluoromethylphenyl)-
1H-tetrazole [Activator 42]; 15 min; ∼95%/∼89%/∼99%). Suita-
ble activators were identified through initial screening of common
activators (results not discussed). The studied 9-mer sequences have
been previously used to study ONs modified with 20-N-(pyren-1-yl)-
methyl-20-amino-R-L-LNA thymine monomer L.9b ONs containing
a single incorporation in the 50-GBG ATA TGC context are denoted
V1, W1, Y1, Z1, Q1, S1, V1 and L1, respectively. Similar conventions
apply for ONs in the B2ꢀB7 series (Table 1). In addition, the
following descriptive nomenclature is used: O20-Nap (W series),
O20-Py (X series), O20-PyMe (Y series), O20-CorMe (Z series),
N20-PyMe (Q series), N20-PyCO (S series), N20-PyAc (V series),
and N20-PyMe-R-L-LNA (L series). Reference DNA and RNA
strands are denoted D1/D2 and R1/R2, respectively.
The thermal affinity of B1ꢀB7 toward cDNA or cRNA targets
was evaluated via UV thermal denaturation experiments using a
medium salt buffer that mimics physiological ionic strengths
([Na+] = 110 mM, Tables 1 and 2). As expected, all denaturation
curves display monophasic sigmoidal transitions (Figures S1 and
S2 in Supporting Information). Changes in thermal denaturation
temperatures (Tm values) of modified duplexes are discussed
relative to Tm values of unmodified reference duplexes, unless
otherwise mentioned. The effect on duplex thermostability
upon exchanging thymine moieties (reference ONs) with uracil
moieties (modified ONs) is recognized (ΔTm/modification
[ΔTm/mod] ≈ ꢀ0.5 °C)29 but otherwise disregarded.
Thermal Affinity toward Complementary DNA/RNA;
O20-Intercalator-Functionalized Uridine Monomers WꢀZ.
The effects on thermal DNA affinity upon incorporation of O20-
functionalized uridine monomers WꢀZ into ONs vary drama-
tically (ΔTm/mod = ꢀ13.0 to +20.0 °C, Table 1). The observed
trends in DNA affinity of singly modified ONs (Z g L > Y .
X . V) suggest that (a) 20-O-(pyren-1-yl)methyluridine mono-
mer Y is a mimic of 20-N-(pyren-1-yl)methyl-20-amino-R-L-LNA
benchmark monomer L with respect to DNA-hybridization
properties and binding mode, albeit slightly lower duplex ther-
mostabilization is observed (ΔTm/mod = +3.5 to +12.5 °C vs
+6.5 to +15.5 °C, for Y1ꢀY5 and L1ꢀL5, respectively, Table 1);
(b) increasing the intercalator surface area leads to additional
stabilization of DNA duplexes (O20-PyMe monomer Y f O20-
CorMe monomer Z, ΔTm/mod = +4.5 to +20.0 °C, Table 1); (c)
shortening the linker between the furanose and pyrene moiety by
one CH2-group results in markedly lower thermal affinity toward
cDNA (O20-PyMe monomer Y f O20-Py monomer X,
ΔTm/mod = ꢀ3.5 to +4.0 °C, Table 1); and (d) concomitant
reduction in aromatic surface area results in additionally
decreased DNA duplex thermostability (O20-Py monomer
X f O20-Nap monomer W, ΔTm/mod = ꢀ13.0 to ꢀ5.0 °C,
Table 1). The results underline that intercalation is an
important binding mode for monomers Y and Z and corro-
borate earlier studies demonstrating that pyrene and coronene
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Table 1. Tm Values of Duplexes between B1ꢀB7 and Complementary DNA Targetsa
Tm [ΔTm/mod] (°C)
ON
duplex
B =
T
W
X
Y
Z
Q
S
V
L9b
B1 50-GBG ATA TGC
D2 30-CAC TAT ACG
29.5 21.5 [ꢀ8.0] 26.5 [ꢀ3.0] 34.5 [+5.0] 34.0 [+4.5] 34.5 [+5.0] 23.5 [ꢀ6.0] 29.0 [ꢀ0.5] 36.5 [+7.0]
29.5 24.5 [ꢀ5.0] 33.5 [+4.0] 42.0 [+12.5] 49.5 [+20.0] 43.5 [+14.0] 32.5 [+3.0] 35.5 [+6.0] 43.5 [+14.0]
B2 50-GTG ABA TGC
D2 30-CAC TAT ACG
B3 50-GTG ATA BGC
D2 30-CAC TAT ACG
29.5 24.5 [ꢀ5.0] 26.0 [ꢀ3.5] 37.5 [+8.0] 40.5 [+11.0] ND
ND
ND
40.0 [+10.5]
D1 50-GTG ATA TGC
B4 30-CAC BAT ACG
29.5 16.5 [ꢀ13.0] 26.0 [ꢀ3.5] 33.0 [+3.5] 36.0 [+6.5] 31.0 [+1.5] 23.5 [ꢀ6.0] 30.5 [+1.0] 36.0 [+6.5]
29.5 24.5 [ꢀ5.0] 30.5 [+1.0] 41.0 [+11.5] 45.5 [+16.0] 42.5 [+13.0] 33.5 [+4.0] 36.0 [+6.5] 45.0 [+15.5]
D1 50-GTG ATA TGC
B5 30-CAC TAB ACG
D1 50-GTG ATA TGC
B6 30-CAC BAB ACG
29.5 ND
25.5 [ꢀ2.0] 43.5 [+7.0] 47.0 [+8.8] 43.5 [+7.0] ND
37.0 [+3.8] ND
B7 50-GBG ABA BGC
D2 30-CAC TAT ACG
29.5 ND
25.5 [ꢀ1.3] 51.0 [+7.2] ND
ND
ND
ND
ND
a ΔTm = change in Tm's relative to unmodified reference duplex; Tm's determined as the maximum of the first derivative of melting curves (A260 vs T)
recorded in medium salt buffer ([Na+] = 110 mM, [Clꢀ] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 μM of each strand. Tm's are averages
of at least two measurements within 1.0 °C; A = adenin-9-yl DNA monomer, C = cytosin-1-yl DNA monomer, G = guanin-9-yl DNA monomer,
T = thymin-1-yl DNA monomer. ND = not determined. For structures of monomers QꢀZ see Figure 1.
moieties engage in efficient πꢀπ stacking with flanking nucleo-
bases.16,30,31 The shorter linker of monomers W and X,
in contrast, seems to render intercalative binding modes less
likely. The magnitude of DNA duplex thermostabilization is
highly sequence-dependent, in particular for ONs modified
with monomer Z. Particularly pronounced stabilization is
observed when Z is centrally incorporated and flanked by
purines (e.g., compare ΔTm/mod values for Z2 and Z4). This
reflects strong πꢀπ stacking efficiency with large 30-flanking
purines.9b,14
Singly modified ONs B1ꢀB5 display similar relative trends in
thermal affinity toward cRNA (Z ≈ L > Y . X > W) as toward
cDNA except that the resulting duplexes are considerably less
stable (ΔTm/mod = ꢀ12.0 to +10.5 °C, Table 2). In fact, none of
the monomers consistently induces increased thermal affinity
relative to reference strand D1. Substantial DNA-selectivity,
defined as ΔΔTm (DNA-RNA) = ΔTm (vs DNA) ꢀ ΔTm (vs
RNA) > 0 °C, is therefore observed (Table 3). The DNA-
selectivity of O20-CH2Py-modified Y1ꢀY5 and O20-CH2Cor-
modified Z1ꢀZ5 is especially pronounced and comparable
to that of the corresponding N20-PyMe-R-L-LNA L1ꢀL5
(Table 3). The extensive DNA-selectivity of doubly O20-
CH2Cor-modified Z6 is particularly noteworthy and hints at
interesting DNA-targeting applications for multilabeled ONs
(ΔΔTm (DNA-RNA) = 31.0 °C, Table 3). DNA-selective hybri-
dization is typically observed for ONs modified with interca-
lating moieties,9b,14,32,33 since intercalation favors the less
compressed B-type helix geometry of DNA:DNA duplexes.
In contrast, most of the O20-Py-modified X-series and all
O20-Nap-modified W1ꢀW5, in particular, display much lower
DNA-selectivity, indicating that intercalative binding modes are
less prominent with these monomers than with Y and Z mono-
mers (Table 3).
Thermal Affinity toward Complementary DNA/RNA; N20-
Pyrene-Functionalized 20-N-Methyl-20-aminodeoxyuridine
Monomers Q, S, and V. The highly variable effects on thermal
affinity toward DNA targets upon incorporation of N20-pyrene-
functionalized 20-N-methyl-20-aminodeoxyuridine monomers Q,
S, and V into ONs are intriguing considering the very conserva-
tive differences in linker chemistry and linker length (ΔTm/
mod = ꢀ6.0 to +14.0 °C, Table 1). The observed trends in DNA
duplex thermostabilization of singly modified ONs (L > Q .
V > S) suggest that (a) 20-N-(pyren-1-ylmethyl)-20-N-methyla-
minouridine monomer Q indeed is a mimic of N20-PyMe-R-L-
LNA monomer L with respect to DNA-hybridization properties
and binding mode (ΔTm/mod = +1.5 to +14.0 °C, Table 1); (b)
monomers where the pyrene moiety is attached via N20-alkyl
linkers are preferred over those with N20-alkanoyl linkers
(compare ΔTm/mod for S1ꢀS5 = ꢀ6.0 to +4.0 °C with data
for Q1ꢀQ5, Table 1); and (c) extending the N20-alkanoyl linker
between the furanose and pyrene moiety as in ONs modified
with 20-N-(pyren-1-ylmethycarbonyl)-20-aminouridine monomer
V, partially reverses the detrimental effects of N20-acylation on
DNA duplex thermostability (N20-PyCO monomer S f N20-
PyAc monomer V, ΔTm/mod = ꢀ0.5 to +6.5 °C, Table 1). ONs
with central incorporations of N20-acylated 20-N-aminouridine
monomers (i.e., without the 20-N-methyl group of monomers
Q, S, and V) are known to display low affinity toward DNA
targets.34,35 This suggests that the low thermal affinity ob-
served for ONs in the S and V series is a consequence of the 20-
N-amido substituent rather than the "extra" 20-N-methyl group.
We speculate that the rigid 20-N-alkanoyl linker positions the
intercalator in a position less favorable for affinity-enhancing
intercalation and/or that increased solvation of the linker stabilizes
the single-stranded state rendering hybridization less energetically
favorable.
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Table 2. Tm Values of Duplexes between B1ꢀB7 and Complementary RNA Targetsa
Tm [ΔTm/mod] (°C)
ON
duplex
B =
T
W
X
Y
Z
Q
S
V
L9b
B1 50-GBG ATA TGC
R2 30-CAC UAU ACG
26.5 21.0 [ꢀ5.5] 22.5 [ꢀ4.0] 24.5 [ꢀ2.0] 21.5 [ꢀ5.0] 22.0 [ꢀ4.5] 14.0 [ꢀ12.5] 20.0 [ꢀ6.5] 27.0 [+0.5]
26.5 16.0 [ꢀ10.5] 22.5 [ꢀ4.0] 30.5 [+4.0] 37.0 [+10.5] 29.0 [+2.5] 18.0 [ꢀ8.5] 28.0 [+1.5] 31.5 [+5.0]
B2 50-GTG ABA TGC
R2 30-CAC UAU ACG
B3 50-GTG ATA BGC
R2 30-CAC UAU ACG
26.5 18.0 [ꢀ8.5] 14.5 [ꢀ12.0] 26.5 [(0.0] 30.5 [+4.0] ND
ND
ND
28.0 [+1.5]
R1 50-GUG AUA UGC
B4 30-CAC BAT ACG
24.5 16.5 [ꢀ8.0] 16.5 [ꢀ8.0] 20.0 [ꢀ4.5] 22.5 [ꢀ2.0] 17.5 [ꢀ7.0] 9.0 [ꢀ15.5] 16.0 [ꢀ8.5] 23.5 [ꢀ1.0]
24.5 19.5 [ꢀ5.0] 21.5 [ꢀ3.0] 27.0 [+2.5] 30.5 [+6.0] 27.0 [+2.5] 16.0 [ꢀ8.5] 26.5 [+2.0] 32.0 [+7.5]
R1 50-GUG AUA UGC
B5 30-CAC TAB ACG
R1 50-GUG AUA UGC
B6 30-CAC BAB ACG
24.5 ND
14.5 [ꢀ5.0] 24.0 [ꢀ0.3] 11.0 [ꢀ6.8] 20.0 [ꢀ2.3] ND
19.0 [ꢀ2.8] ND
B7 50-GBG ABA BGC
D2 30-CAC UAU ACG
26.5 ND
15.5 [ꢀ3.7] 27.5 [+0.3] ND
ND
ND
ND
ND
a For conditions of thermal denaturation experiments, see Table 1.
Table 3. DNA-Selectivity of B1ꢀB7a
binding mode for monomer S but that it interferes with duplex
formation. The less pronounced DNA-selectivity of V1ꢀV6
seems to suggest that intercalative pyrene binding modes are less
dominant for monomer V.
ΔΔTm (DNA-RNA) [°C]
ON
sequence
B =
W
X
Y
Z
Q
S
V
L9b
To sum up, the thermal denaturation data suggest that O20-
PyMe monomer Y, O20-CorMe monomer Z, and N20-PyMe
monomer Q are mimics of N20-PyMe-R-L-LNA monomer L and
attractive candidates for DNA targeting applications.
B1 50-GBG ATA TGC
B2 50-GTG ABA TGC
B3 50-GTG ATA BGC
B4 30-CAC BAT ACG
B5 30-CAC TAB ACG
B6 30-CAC BAB ACG
B7 50-GBG ABA BGC
ꢀ2.5 +1.0 +7.0 +9.5 +9.5 +6.5 +6.0 +6.5
+5.5 +8.0 +8.5 +9.5 +11.5 +11.5 +4.5 +9.0
+3.5 +8.5 +8.0 +7.0 ND ND ND +9.0
+5.0 +4.5 +8.0 +8.5 +8.5 +9.5 +9.5 +7.5
Mismatch Discrimination. The WatsonꢀCrick specificity
of singly modified ONs (B2 series) was evaluated using DNA
(Table 4) or RNA targets (Table S4 in Supporting Information)
with mismatched nucleotides opposite of the incorporation site.
ONs in the B2 series generally display reduced DNA target
specificity relative to reference strand D1, which is in agreement
with observations on other intercalator-functionalized ONs.9b,36,37
For example, O20-PyMe-modified Y2 and O20-CorMe-modified
Z2 discriminate dT-mismatches very poorly relative to D1
(compare ΔTm values for D1, Y2, and Z2 against targets with
a dT-mismatch, Table 4), while dC- and dG-mismatches almost
are as efficiently discriminated as with D1. N20-PyMe-R-L-LNA
L2 has a similar specificity profile but discriminates the dT-
mismatch even more poorly. Interestingly, N20-PyMe-modified
Q2 displays substantially better discrimination of dC- and dT-
mismatches than Y2, Z2, and L2 rendering it as the most specific
of the high-affinity DNA-targeting modifications. Even minor
changes in linker chemistry and length have marked influence
on target specificity. Thus, N20-PyCO-modified S2 displays
similar DNA target specificity as Y2, Z2, and L2, while N20-
PyAc-modified V2 discriminates DNA mismatches more
poorly. Similarly, O20-Nap-modified W2 exhibits very poor
target specificity, while O20-Py-modified X2 displays much high-
er target specificity than W2 or O20-PyMe-modified Y2
(Table 4). Structural models (e.g., NMR solution structures)
will be necessary to fully understand the molecular underpin-
nings of these trends.
0
+4.0 +9.0 +10.0 +10.5 +12.5 +4.5 +8.0
ND +6.0 +14.5 +31.0 +18.5 ND +13.0 ND
ND +7.0 +20.5 ND ND ND ND ND
a DNA-selectivity defined as ΔΔTm (DNA-RNA) = ΔTm (vs DNA) ꢀ
ΔTm (vs RNA).
The hybridization characteristics of ONs modified with mono-
mers Q/S/V share several points of similarity with ONs modified
with monomers WꢀZ: (a) thermostabilization of DNA duplexes
is highly sequence-dependent; (b) incorporation of two mono-
mers as next-nearest neighbors leads to additive increases in
DNA duplex thermostability (i.e., compare ΔTm/mod values for
Q4ꢀQ6, Table 1); (c) duplexes with complementary RNA are
dramatically destabilized (ΔTm/mod = ꢀ15.5 to +2.5 °C,
Table 2); and (d) extensive DNA-selectivity is observed which
is most pronounced for N20-PyMe-modified Q1ꢀQ5 and N20-
PyCO-modified S1ꢀS5 (ΔΔTm (DNA-RNA) up to +12.5 °C,
Table 3). The latter is in agreement with the suggested inter-
calative pyrene binding mode for monomer Q,15 while the
pronounced DNA-selectivity of S1ꢀS5 is more surprising
given the low DNA duplex thermostability. This indicates
that intercalation of the pyrene moiety remains an important
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Table 4. Discrimination of Mismatched DNA Targets by B2
Series and Reference Strandsa
be used as general motifs to develop ONs with improved target
specificity.
Optical Spectroscopy. Absorption and steady state fluores-
cence emission spectra of ONs modified with monomers Q, S, V,
X, and Y in the presence or absence of cDNA/cRNA targets were
recorded to gain additional insight into the binding modes of the
pyrene moieties. Intercalation of pyrene moieties is known to
induce bathochromic shifts of pyrene absorption peaks.38 In
agreement with this, ONs modified with monomers Q, S, V, and
Y display significant bathochromic shifts upon hybridization
with DNA targets (average Δλmax = 2.8ꢀ5.0 nm, respectively,
Table 6; see also Figure S4 and Tables S5 and S6 in Supporting
Information). Hybridization with cRNA leads to slightly
smaller bathochromic shifts as intercalation into increasingly
DNA: 30-CAC TBT ACG
T
m [°C]
ΔTm [°C]
ON
sequence
B =
A
C
G
T
D1
W2
X2
Y2
Z2
Q2
S2
50-GTG ATA TGC
50-GTG AWA TGC
50-GTG AXA TGC
50-GTG AYA TGC
50-GTG AZA TGC
50-GTG AQA TGC
50-GTG ASA TGC
50-GTG AVA TGC
29.5
24.5
33.5
42.0
49.0
43.5
32.5
35.5
43.5
ꢀ16.5 ꢀ9.5 ꢀ17.0
ꢀ11.0 +2.0 ꢀ3.5
ꢀ23.5 ꢀ7.0 ꢀ13.0
ꢀ13.0 ꢀ5.0
ꢀ13.5 ꢀ6.0
ꢀ6.5
ꢀ7.0
ꢀ22.0 ꢀ3.5 ꢀ12.0
compressed A-type duplexes is less favorable (average Δλmax
=
ꢀ11.5 ꢀ9.0
ꢀ11.5 +1.5
ꢀ12.5 ꢀ5.5
ꢀ8.5
ꢀ3.5
ꢀ3.5
2.0ꢀ4.5 nm, Table 6). O20-Py-modified X1ꢀX5 display
distinctly smaller hybridization-induced bathochromic shifts
than the other pyrene-functionalized ONs (average Δλmax
∼0.6 nm, Table 6), which substantiates the trends from
thermal denaturation studies suggesting that intercalation
of the pyrene moiety is a less important binding mode for
monomer X.
V2
L29b 50-GTG ALA TGC
a For conditions of thermal denaturation experiments, see Table 1. Tm
values of fully matched duplexes are shown in bold. ΔTm = change in Tm
relative to fully matched DNA:DNA duplex.
The steady-state fluorescence emission spectra (λex = 350 nm;
λem = 360ꢀ600 nm; T = 5 °C) display the two expected vibronic
bands I and III at λem = 382 ( 3 and 402 ( 3 nm, respectively, as
well as a small shoulder at ∼420 nm (Figure 2). Hybridization of
ONs modified with monomers Q, S, V, X, or Y with cRNA, and
cDNA in particular, results in reduced fluorescence emission
intensity (see Figure 2 for B4 series; see Figures S5ꢀS9 in
Supporting Information for all series). This trend corroborates
pyrene intercalation as nucleobase moieties are known to
quench pyrene fluorescence via photoinduced electron trans-
fer (PET) with guanine and cytosine moieties being the
strongest quenchers.39 In agreement with this, duplexes invol-
ving the Y, Q, and V series (intercalation is important binding
mode) display far lower emission intensity than those invol-
ving the X series (intercalation is less important binding
mode). Sequence-specific deviations from general trends are
observed in some cases (e.g., Q2, S1, S4, and S5, see Figures
S7ꢀS8 in Supporting Information), which likely reflects a
trend-defying duplex geometry or positioning of the pyrene
moiety. For additional discussion of fluorescence emission
data, please see the Supporting Information.
Table 5. Discrimination of Mismatched DNA Targets by B6
Series and Reference Strandsa
DNA: 50-GTG ABA ACG
T
m [°C]
ΔTm [°C]
ON
sequence
B =
T
A
C
G
D2 30-CAC TAT ACG
29.5
25.5
43.5
47.0
43.5
37.0
ꢀ17.0 ꢀ15.5
ꢀ9.0
X6
Y6
Z6
30-CAC XAX ACG
30-CAC YAY ACG
30-CAC ZAZ ACG
ꢀ15.5 ꢀ14.0 ꢀ14.5
ꢀ24.0 ꢀ17.0 ꢀ14.0
ꢀ19.5 ꢀ13.0 ꢀ11.0
ꢀ21.5 ꢀ10.5 ꢀ13.5
ꢀ14.5 ꢀ13.5 ꢀ11.0
Q6 30-CAC QAQ ACG
V6
30-CAC VAV ACG
a For conditions of thermal denaturation experiments, see Table 1. Tm
values of fully matched duplexes are shown in bold. ΔTm = change in Tm
relative to fully matched DNA:DNA duplex.
The specificity of ONs with two modifications positioned as
next-nearest neighbors (B6 series) was evaluated against DNA
targets with a single central mismatched nucleotide opposite the
central 20-deoxyadenosine residue (Table 5). This probe design
generally results in improved mismatch discrimination relative to
reference strand D2 with specificity decreasing in the following
manner: Y6 > X6 > Z6 ≈ Q6 > V6; A- and G-mismatches are
particularly efficiently discriminated. Subsequent experiments
will reveal if these “intercalator-capped fidelity cassettes” can
On balance, the data from the thermal denaturation and
optical spectroscopy studies (DNA-selectivity; mismatch discri-
mination; UVꢀvis; fluorescence) suggest that intercalation of
the attached hydrocarbon is a possible binding mode for all
studied monomers. Intercalation is least important for mono-
mers W and X, while being a dominant binding mode for
monomers Y, Z, and Q.
Table 6. Average Bathochromic Shifts of Pyrene Absorption Bands (λem ∼350 nm) upon Hybridization of Pyrene-Functionalized
ONs with Complementary DNA/RNA Targetsa
average Δλmax (nm)
B =
X
Y
Q
S
V
L9b
+DNA
+RNA
+DNA
+RNA
+DNA
+RNA
+DNA
+RNA
+DNA
+RNA
+DNA
+RNA
0.6 ( 1.8 0.6 ( 1.3 3.4 ( 1.1 2.6 ( 0.9 5.0 ( 0.8 2.3 ( 1.7 2.8 ( 1.0 2.0 ( 1.8 4.5 ( 0.6 4.5 ( 1.3 2.5 ( 1.0 1.0 ( 0.8
a Values are averages of measurements performed for B1ꢀB5 at 5 °C (Q, S, V), 7 °C (X, Y), or room temperature (L).9b Buffer conditions are as for
thermal denaturation experiments. ( denotes standard deviation. For full data set, see Tables S5 and S6 in Supporting Information.
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Figure 2. Fluorescence emission spectra of B4 series in the presence or absence of cDNA/RNA (λex = 350 nm; T = 5 °C; [ON] = 1.0 μM). Note that
different y-axes are used.
’ CONCLUSION
We have developed short routes to a series of
O20-intercalator-functionalized uridine and N20-intercalator-
functionalized 20-N-methyl-20-aminouridine phosphoramidites.
Thermal denaturation studies of correspondingly modified ONs
reveal that even very conservative changes in linker chemistry,
linker length, or surface area of the intercalator translate into
markedly different DNA-hybridization characteristics (Figure 3).
We propose the following guidelines for design of high-affinity
DNA-targeting monomers based on uridine or 20-N-methyl-20-
aminouridine scaffolds: (a) direct attachment of intercalators to
the O20-position of uridine should be avoided (e.g., monomer W
and X), (b) attachment of intercalators to the N20-position of
2-N-methyl-20-aminouridine via alkanoyl linkers should be
Figure 3. Qualitative representation of DNA-hybridization character-
istics of O20-intercalator-functionalized uridine (light gray) and N20-
intercalator-functionalized 20-N-methyl-20-aminouridine (dark gray)
monomers studied herein relative to reference monomers L and T
(black).
avoided (e.g., monomer S and V), and (c) attachment of large
intercalators via a methylene linker is desirable (e.g., mono-
mers Q, Y, and Z). Thus, ONs modified with 20-O-(pyren-1-yl)-
methyluridine monomer Y, 20-O-(coronen-1-yl)methyluridine
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monomer Z, or 20-N-(pyren-1-ylmethyl)-20-N-methylaminouridine
monomer Q display similar thermal affinity toward DNA com-
plements (average ΔTm/mod between +8 and +12 °C) and
higher target specificity than ONs modified with 20-N-(pyren-1-yl)-
methyl-20-amino-R-L-LNA benchmark monomer L (Figure 3).
The highly DNA-selective hybridization of these ONs, and of the
doubly O20-CH2Cor-modified Z6 in particular (ΔΔTm (DNA-
RNA) = 31.0 °C), hints at interesting applications within DNA
diagnostics. Straightforward access to these monomers will likely
spur their use within nucleic acid based diagnostics, therapeutics,
and material science.10ꢀ12,40ꢀ42 Studies along these lines are
ongoing and will be reported shortly.
155.4, 150.5, 140.4 (C6), 133.9, 129.2 (Nap), 128.7, 127.4 (Nap), 126.6
(Nap), 126.4 (Nap), 123.8 (Nap), 118.9 (Nap), 108.9 (Nap), 102.0
(C5), 86.4 (C10), 85.2 (C40), 79.3 (C20), 68.2 (C30), 60.4 (C50).
Although 2W is obtained in lower yield than via a recently published
microwave-assisted approach (∼50% yield), the current approach does
not require access to microwave reactors. The 1H NMR data for 2W are
in agreement with the literature report.18
20-O-(Pyren-1-yl)uridine (2X). A mixture of 2,20-anhydrouridine
1 (0.30 g, 1.33 mmol) and 1-pyrenol19 (0.86 g, 3.97 mmol) was reacted
and purified as described above to afford nucleoside 2X (0.26 g, 44%) as
a pale yellow solid. Rf 0.4 (10% MeOH in CH2Cl2, v/v); MALDI-HRMS
1
m/z 467.1217 ([M + Na]+, C25H20N2O6 Na+, calcd 467.1214); H
3
NMR (DMSO-d6) δ 11.36 (s, 1H, ex, NH), 8.45 (d, 1H, J = 9.3 Hz, Py),
8.20ꢀ8.24 (m, 3H, Py), 8.14 (d, 1H, J = 9.3 Hz, Py), 7.99ꢀ8.09 (m, 4H,
H6, Py), 7.86 (1H, d, J = 8.5 Hz, Py), 6.35 (d, 1H, J = 4.6 Hz, H10), 5.68
(d, 1H, J = 8.2 Hz, H5), 5.63 (br s, 1H, ex, 30-OH), 5.30 (br s, 1H, ex,
50-OH), 5.22ꢀ5.25 (ap t, 1H, H20), 4.55ꢀ4.56 (m, 1H, H30), 4.19ꢀ4.21
(m, 1H, H40), 3.81ꢀ3.84 (ap d, 1H, H50), 3.72ꢀ3.75 (ap d, 1H, H50);
13C NMR (DMSO-d6) δ 162.9, 151.7, 150.5, 140.3 (C6), 131.0, 130.9,
127.1 (Py), 126.4 (Py), 125.6 (Py), 125.2, 125.0 (Py), 124.8 (Py), 124.5
(Py), 124.3 (Py), 123.9, 121.1 (Py), 120.1, 111.9 (Py), 102.0 (C5), 86.6
(C10), 85.4 (C40), 80.9 (C20), 68.5 (C30), 60.4 (C50).
’ EXPERIMENTAL SECTION
General Experimental Section. Unless otherwise noted, re-
agents and solvents were commercially available, of analytical grade,
and used without further purification. Petroleum ether of the distillation
range 60ꢀ80 °C was used. Solvents were dried over activated molecular
sieves: acetonitrile and THF (3 Å); CH2Cl2, 1,2-dichloroethane, N,
N0-diisopropylethylamine, and anhydrous DMSO (4 Å). Water content
of “anhydrous” solvents was verified on Karl Fischer apparatus. Reac-
tions were conducted under argon whenever anhydrous solvents were
used. Reactions were monitored by TLC using silica gel coated plates
with a fluorescence indicator (SiO2-60, F-254) which were visualized
(a) under UV light and/or (b) by dipping in 5% conc H2SO4 in absolute
ethanol (v/v) followed by heating. Silica gel column chromatography
was performed with silica gel 60 (particle size 0.040ꢀ0.063 mm) using
moderate pressure (pressure ball). Evaporation of solvents was carried
out under reduced pressure at temperatures below 45 °C. After column
chromatography, appropriate fractions were pooled, evaporated, and
dried at high vacuum for at least 12 h to give the obtained products in
high purity (>95%) as ascertained by 1D NMR techniques. Chemical
General Protocol for Coupling between 1 and Arylmethyl
Alcohol (ArCH2OH) for Preparation of 2Y/2Z (Description for
∼44.2 mmol Scale). The appropriate aromatic alcohol (ArCH2OH),
NaHCO3, and 1.0 M BH3 in THF were placed in a pressure tube,
suspended in anhydrous DMSO, and stirred under an argon atmosphere
at rt until effervescence ceased (∼10 min). At this point, 2,20-anhy-
drouridine 120 was added (specific quantities of substrates and reagents
given below), the pressure tube was purged with argon and sealed, and
the reaction was heated at ∼160 °C until analytical TLC indicated full
conversion (∼3 h). At this point, the reaction mixture was poured into
water (200 mL), stirred for 30 min, and diluted with EtOAc (500 mL).
The organic phase was washed with water (4 ꢁ 200 mL) and evaporated
to dryness, and the resulting crude was purified by silica gel column
chromatography (2ꢀ4%, MeOH in CH2Cl2, v/v) to afford a residue,
which was precipitated from cold acetone to obtain nucleoside 2 (yields
specified below).
1
shifts of H NMR (500 MHz), 13C NMR (125.6 MHz), and/or 31P
NMR (121.5 MHz) are reported relative to deuterated solvent or other
internal standards (80% phosphoric acid for 31P NMR). Exchangeable
(ex) protons were detected by disappearance of signals upon D2O
addition. Assignments of NMR spectra are based on 2D spectra (HSQC,
COSY) and DEPT spectra. Quaternary carbons are not assigned in 13C
NMR but verified from HSQC and DEPT spectra (absence of signals).
MALDI-HRMS spectra of compounds were recorded on a Q-TOF mass
spectrometer using 2,5-dihydroxybenzoic acid (DHB) as a matrix and
poly(ethylene glycol) (PEG 600) as an internal calibration standard.
General Protocol for Coupling between 1 and Phenols
(ArOH) ToPrepare 2W/2X (Descriptionfor∼1.33 mmol Scale).
The appropriate phenol and 2,20-anhydrouridine 117 were placed in a
sealed pressure tube (specific quantities of substrates and reagents given
below) and heated (165 °C for 2W; 175 °C for 2X) until analytical TLC
indicated full conversion (∼2 h). The resulting crude was purified by
silica gel column chromatography (2ꢀ4% MeOH in CH2Cl2, v/v) to
afford nucleoside 2W/2X (yields specified below).
20-O-(Pyren-1-yl-methyl)uridine (2Y). 2,20-Anhydrouridine 1
(10.00 g, 44.2 mmol), pyren-1-ylmethanol23 (20.5 g, 88.4 mmol),
NaHCO3 (0.73 g. 8.80 mmol), and 1.0 M BH3 in THF (24.5 mL,
22.0 mmol) and anhydrous DMSO (40 mL) were mixed, reacted, worked
up, and purified as described above to afford nucleoside 2Y (5.04 g, 25%)
as a white solid. Rf 0.4 (10% MeOH in CH2Cl2, v/v); MALDI-HRMS
m/z 458.1480 ([M]+, C26H22N2O6 , calcd 458.1472); 1H NMR
+
(DMSO-d6) δ 11.29 (s, 1H, ex, NH), 8.37ꢀ8.39 (d, 1H, J = 9.3 Hz,
Py), 8.29ꢀ8.31 (m, 2H, Py), 8.24ꢀ8.26 (d, 1H, J = 7.7 Hz, Py),
8.17ꢀ8.19 (m, 3H, Py), 8.06ꢀ8.12 (m, 2H, Py), 7.82 (d, 1H, J = 8.4 Hz,
H6), 6.04 (d, 1H, J = 5.1 Hz, H10), 5.43ꢀ5.50 (m, 2H, H5, CH2Py), 5.37
(d, 1H, ex, J = 5.7 Hz, 30-OH), 5.28ꢀ5.30 (d, 1H, J = 12.0 Hz, CH2Py),
5.11 (t, 1H, ex, J = 4.9 Hz, 50-OH), 4.26ꢀ4.31 (m, 1H, H30), 4.18ꢀ4.21
(m, 1H, H20), 3.96ꢀ3.97 (m, 1H, H40), 3.64ꢀ3.68 (m, 1H, H50),
3.59ꢀ3.62 (m, 1H, H50); 13C NMR (DMSO-d6) δ 162.9, 150.6, 140.1
(C6), 131.4, 130.7, 130.2, 128.7, 127.4 (Py), 127.3 (Py), 127.0 (Py),
126.2 (Py), 125.3 (Py), 124.5 (Py), 124.0 (Py), 123.8, 123.5 (Py), 101.7
(C5), 86.2 (C10), 85.4 (C40), 80.9 (C20), 69.8 (CH2Py), 68.5 (C30),
60.6 (C50). 1H NMR data for nucleoside 2Y are in agreement with data
where nucleoside 2Y was obtained via a different route.17 Full experi-
mental details on the preparation and characterization of 2Y have, to our
knowledge, not been previously published.
20-O-(Napth-2-yl)uridine (2W). A mixture of 2,20-anhydrouri-
dine 1 (1.00 g, 4.42 mmol) and 2-napthol (2.40 g, 22.1 mmol) was
reacted and purified as described above to afford nucleoside 2W (0.41 g,
25%) as a light brown solid. Rf 0.4 (10% MeOH in CH2Cl2, v/v);
MALDI-HRMS m/z 393.1039 ([M + Na]+, C19H18N2O6 Na+, calcd
3
393.1057); 1H NMR (DMSO-d6) δ 11.34 (s, 1H, ex, NH), 8.03 (1H, d,
J = 8.1 Hz, H6), 7.82ꢀ7.85 (ap d, 2H, Nap), 7.73ꢀ7.75 (1H, d, J = 8.3
Hz, Nap), 7.44ꢀ7.48 (ap t, 1H, Nap), 7.41 (d, 1H, J = 2.5 Hz, Nap),
7.34ꢀ7.37 (ap t, 1H, Nap), 7.23ꢀ7.25 (dd, 1H, J = 9.1 Hz, J = 2.5 Hz,
Nap), 6.14 (d, 1H, J = 4.9 Hz, H10), 5.68 (d, 1H, J = 8.1 Hz, H5), 5.46 (d,
1H, ex, J = 6.4 Hz, 30-OH), 5.25 (t, 1H, ex, J = 5.2 Hz, 50-OH), 5.02 (ap t,
1H, H20), 4.43ꢀ4.46 (m, 1H, H30), 4.02ꢀ4.05 (m, 1H, H40), 3.74ꢀ3.78
(m, 1H, H50), 3.65ꢀ3.69 (m, 1H, H50); 13C NMR (DMSO-d6) δ 162.9,
20-O-(Coronen-1-yl-methyl)uridine (2Z). 2,20-Anhydrouridine
1 (1.40 g, 6.19 mmol), coronen-1-ylmethanol24 (4.08 g, 12.4 mmol),
NaHCO3 (0.104 g. 1.24 mmol), and 1.0 M BH3 in THF (3.5 mL,
3.1 mmol) and anhydrous DMSO (40 mL) were mixed, reacted, worked
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up, and purified as described above with a minor modification. The
precipitate that formed upon pouring the reaction mixture into water
was collected by filtration, washed with water (3 ꢁ 100 mL), and
purified by column chromatography to afford nucleoside 2Z (450 mg,
13%) as a pale yellow solid. Rf 0.4 (10% MeOH in CH2Cl2, v/v);
4.61ꢀ4.65 (m, 1H, H30), 4.34ꢀ4.37 (m, 1H, H40), 3.74 (s, 6H, 2 ꢁ
CH3O), 3.46ꢀ3.50 (m, 1H, H50), 3.38ꢀ3.41 (m, 1H, H50); 13C NMR
(DMSO-d6) δ 162.9, 158.1, 151.7, 150.3, 144.6, 140.5 (C6), 135.4, 135.1,
131.1, 131.0, 129.78 (DMTr), 129.76 (DMTr), 127.9 (DMTr), 127.1 (Py),
126.8 (DMTr), 126.4 (Py), 125.6 (Py), 125.3, 125.1 (Py), 124.9, 124.5
(Py), 124.3 (Py), 124.0, 121.2 (Py), 120.1, 113.2 (DMTr), 112.3 (Py),
101.7 (C5), 87.8 (C10), 85.9, 82.7 (C40), 80.7 (C20), 68.7 (C30), 62.7
(C50), 55.0 (CH3O).
MALDI-HRMS m/z 579.1537 ([M + Na]+, C34H24N2O6 Na+, calcd
3
579.1532); 1H NMR (DMSO-d6) δ 11.30 (br d, ex, 1H, J = 1.9 Hz, NH),
9.13ꢀ9.15 (d, 1H, J = 8.7 Hz, Cor), 8.93ꢀ9.03 (m, 10H, Cor), 7.82 (d,
1H, J = 8.0 Hz, H6), 6.18 (d, 1H, J = 4.9 Hz, H10), 5.85ꢀ5.88 (d, 1H, J =
12.1 Hz, CH2Cor), 5.67ꢀ5.69 (d, 1H, J = 12.1 Hz, CH2Cor), 5.52
(d, 1H, ex, J = 5.5 Hz, 30-OH), 5.38 (dd, 1H, J = 8.0 Hz, 1.9 Hz, H5), 5.11
(ap t, 1H, ex, 50-OH), 4.37ꢀ4.43 (m, 2H, H20, H30), 4.03ꢀ4.06 (m, 1H,
H40), 3.63ꢀ3.71 (m, 2H, H50); 13C NMR (DMSO-d6) δ 162.9, 150.6,
140.1 (C6), 132.1, 128.2, 128.1, 127.9, 127.4, 126.5, 126.23 (Cor),
126.20 (Cor), 126.1 (Cor), 126.0 (Cor), 122.5 (Cor), 121.8, 121.5,
121.4, 121.23, 121.17, 101.7 (C5), 86.3 (C10), 85.5 (C40), 81.1 (C20),
70.7 (CH2Cor), 68.6 (C30), 60.6 (C50).
20-O-(Pyren-1-yl-methyl)-50-O-(4,40-dimethoxytrityl)uridine
(3Y). Nucleoside 2Y (1.02 g, 2.20 mmol), DMTrCl (1.29 g, 3.30 mmol),
and DMAP (∼18 mg) in anhydrous pyridine (20 mL) were mixed, reac-
ted, workedup, and purified as described above to afford 3Y (1.20 g, 72%) as
pale yellow foam. Rf 0.6 (5%, MeOH in CH2Cl2, v/v); MALDI-HRMS
1
m/z 783.2698 ([M + Na]+, C47H40N2O8 Na+, calcd 783.2677); H
3
NMR (DMSO-d6) δ 11.36 (d, 1H, ex, J = 1.9 Hz, NH), 8.41ꢀ8.43
(d, 1H, J = 9.3 Hz, Py), 8.30ꢀ8.32 (m, 2H, Py), 8.14ꢀ8.25 (m, 5H, Py),
8.07ꢀ8.10 (t, 1H, J = 7.7 Hz, Py), 7.63 (d, 1H, J = 8.1 Hz, H6),
7.17ꢀ7.34 (m, 9H, DMTr), 6.82ꢀ6.86 (m, 4H, DMTr), 6.02 (d, 1H, J =
3.9 Hz, H10), 5.48ꢀ5.50 (d, 1H, J = 12.1 Hz, CH2Py), 5.45 (d, 1H, ex, J =
6.3 Hz, 30-OH), 5.36ꢀ5.38 (d, 1H, J = 12.1 Hz, CH2Py), 5.13 (dd, 1H,
J = 8.1 Hz, 1.9 Hz, H5), 4.34ꢀ4.38 (m, 1H, H30), 4.21ꢀ4.24 (m, 1H,
H20), 4.05ꢀ4.09 (m, 1H, H40), 3.71 (s, 3H, CH3O), 3.69 (s, 3H,
CH3O), 3.20ꢀ3.24 (m, 2H, H50); 13C NMR (DMSO-d6) δ 162.8,
158.08, 158.06, 150.4, 144.5, 140.0 (C6), 135.3, 135.0, 131.3, 130.7,
130.2, 129.71 (DMTr), 129.66 (DMTr), 128.7, 127.8 (DMTr), 127.6
(DMTr), 127.4 (Py), 127.3 (Py), 127.0 (Py), 126.7 (DMTr), 126.2
(Py), 125.3 (Py), 124.5 (Py), 124.0, 123.8, 123.4 (Py), 113.20 (DMTr),
113.17 (DMTr), 101.4 (C5), 87.1 (C10), 85.9, 83.1 (C40), 80.6 (C20),
69.9 (CH2Py), 68.7 (C30), 62.8 (C50), 55.0 (CH3O). The protocol and
1H NMR data recorded in CDCl3 for 3Y are in agreement with those
from a related protocol.17 Full experimental details on the preparation
and characterization of 3Y have not been previously reported.
General DMTr-Protection Protocol for the Preparation of
3Wꢀ3Z (Description for ∼2.2 mmol Scale). The appropriate
nucleoside 3 (specific quantities given below) was coevaporated twice
with anhydrous pyridine (15 mL) and redissolved in anhydrous
pyridine. To this were added 4,40-dimethoxytritylchloride (DMTrCl)
and N,N-dimethyl-4-aminopyridine (DMAP), and the reaction mixture
was stirred at rt until TLC indicated complete conversion (∼14 h). The
reaction mixture was diluted with CH2Cl2 (70 mL), and the organic
phase was sequentially washed with water (2 ꢁ 70 mL) and satd aq
NaHCO3 (2 ꢁ 100 mL). The organic phase was evaporated to near
dryness, and the resulting crude coevaporated with absolute EtOH and
toluene (2:1, v/v, 3 ꢁ 6 mL) and purified by silica gel column
chromatography (0ꢀ5%, MeOH in CH2Cl2, v/v) to afford nucleoside
3 (yields specified below).
20-O-(Napth-2-yl)-50-O-(4,40-dimethoxytrityl)uridine (3W).
Nucleoside 2W (150 mg, 0.40 mmol), DMTrCl (240 mg, 0.60 mmol),
and DMAP (∼6 mg) in anhydrous pyridine (7 mL) were mixed, reacted,
worked up, and purified as described above to afford nucleoside 3W
(120 mg, 47%) as a pale yellow foam. Rf 0.6 (5%, MeOH in CH2Cl2,
20-O-(Coronen-1-yl-methyl)-50-O-(4,40-dimethoxytrityl)-
uridine (3Z). Nucleoside 2Z (250 mg, 0.45 mmol), DMTrCl (262 mg,
0.67 mmol), and DMAP (∼15 mg) in anhydrous pyridine (6 mL) were
mixed, reacted, worked up, and purified as described above to afford
nucleoside 3Z (245 mg 63%) as a yellow foam. Rf 0.6 (5%, MeOH in
v/v); MALDI-HRMS m/z 695.2379 ([M + Na]+, C40H36N2O8 Na+,
3
CH2Cl2, v/v); MALDI-HRMS m/z 881.2824 ([M + Na]+, C55H42N2O8
calcd 695.2364); 1H NMR (DMSO-d6) δ 11.40 (d, 1H, ex, J = 2.1 Hz,
NH), 7.83ꢀ7.86 (m, 3H, H6, Nap), 7.68 (d, 1H, J = 8.2 Hz, Nap),
7.24ꢀ7.45 (m, 13H, DMTr, Nap), 6.90ꢀ6.92 (d, 4H, J = 7.1 Hz,
DMTr), 6.06 (d, 1H, J = 3.2 Hz, H10), 5.51 (d, 1H, ex, J = 7.1 Hz, 30-
OH), 5.38ꢀ5.40 (m, 1H, H5), 5.12ꢀ5.14 (m, 1H, H20), 4.51ꢀ4.55 (m,
1H, H30), 4.14ꢀ4.17 (m, 1H, H40), 3.75 (s, 6H, 2 ꢁ CH3O), 3.32ꢀ3.41
(m, 2H, H50); 13C NMR (DMSO-d6) δ 162.9, 158.1, 155.5, 150.3,
144.6, 140.6 (C6), 135.4, 135.2, 133.9, 129.8 (Ar), 129.1 (Nap), 128.8,
127.9 (Ar), 127.7 (Ar), 127.5 (Nap), 126.8 (Ar), 126.6 (Nap), 126.4
(Ar), 123.8 (Ar), 119.0 (Ar), 113.2 (Ar), 109.0 (Ar), 101.8 (C5), 87.6
(C10), 85.9, 82.6 (C40), 79.1 (C20), 68.5 (C30), 62.7 (C50), 55.0 (CH3O).
The described protocol is similar to an independently developed and
recently published protocol; 13C NMR data recorded in CDCl3 are in
agreement with this report.43 NMR spectra recorded in DMSO-d6 have, to
our knowledge, not been provided for this compound.
3
Na+, calcd 881.2839); 1H NMR (DMSO-d6) δ 11.40 (br d, 1H, ex, J =
1.9 Hz, NH), 9.14ꢀ9.16 (d, 1H, J = 8.8 Hz, Cor), 8.96ꢀ9.02 (m, 9H,
Cor), 8.88ꢀ8.90 (d, 1H, J = 8.5 Hz, Cor), 7.63 (d, 1H, J = 8.1 Hz, H6),
7.29ꢀ7.31 (d, 2H, J = 7.4 Hz, DMTr), 7.11ꢀ7.22 (m, 7H, DMTr),
6.74ꢀ6.79 (m, 4H, DMTr), 6.18 (d, 1H, J = 4.1 Hz, H10), 5.87ꢀ5.90 (d,
1H, J = 12.6 Hz, CH2Cor), 5.75ꢀ5.78 (d, 1H, J = 12.6 Hz, CH2Cor),
5.59 (d, 1H, ex, J = 6.3 Hz, 30-OH), 5.06 (dd, 1H, J = 8.1 Hz, 1.9 Hz, H5),
4.46ꢀ4.50 (m, 1H, H30), 4.40ꢀ4.43 (m, 1H, H20), 4.15ꢀ4.18 (m, 1H,
H40), 3.63 (s, 3H, CH3O), 3.58 (s, 3H, CH3O), 3.32ꢀ3.34 (m, 1H,
H50), 3.25ꢀ3.27 (m, 1H, H50); 13C NMR (DMSO-d6) δ 162.8, 158.02,
157.97, 150.4, 144.4, 140.0 (C6), 135.3, 135.0, 132.2, 129.7 (DMTr),
129.6 (DMTr), 128.3, 128.2, 128.0, 127.7 (DMTr), 127.59 (DMTr),
127.56, 126.61 (DMTr), 126.59, 126.41 (Cor), 126.36 (Cor), 126.3
(Cor), 126.23 (Cor), 126.21, 126.19, 126.1 (Cor), 122.6 (Cor), 121.9,
121.6, 121.5, 121.4, 121.35, 121.28, 113.13 (DMTr), 113.09 (DMTr),
101.4 (C5), 87.1 (C10), 85.9, 83.2 (C40), 80.7 (C20), 70.7 (CH2Cor),
68.8 (C30), 62.8 (C50), 54.9 (CH3O), 54.8 (CH3O).
20-O-(Pyren-1-yl)-50-O-(4,40-dimethoxytrityl)uridine (3X). Nu-
cleoside 2X (230 mg, 0.52 mmol), DMTrCl (0.30 g, 0.78 mmol), and
DMAP (∼9 mg) in anhydrous pyridine (8 mL) were mixed, reacted,
worked up, and purified as described above to afford nucleoside 3X (0.30 g,
78%) as a light yellow foam. Rf 0.6 (5%, MeOH in CH2Cl2, v/v); MALDI-
General Phosphitylation Protocol for the Preparation of
4Wꢀ4Z (Description for ∼1 mmol Scale). The appropriate
nucleoside 3 (specific quantities of substrates and reagents given below)
was coevaporated with anhydrous 1,2-dicholoroethane (4 mL) and
redissolved in anhydrous CH2Cl2. To this were added N,N-diisopropy-
lethylamine (DIPEA) and 2-cyanoethyl-N,N-diisopropylchlorophos-
poramidite (PCl reagent), and the reaction mixture was stirred at rt
until TLC indicated complete conversion (∼3 h), whereupon absolute
EtOH (2 mL) and CH2Cl2 (20 mL) were sequentially added to the
HRMS m/z 769.2504 ([M + Na]+, C46H38N2O8 Na+, calcd 769.2520);
3
1H NMR (DMSO-d6) δ 11.32 (s, 1H, ex, NH), 8.49 (d, 1H,
J = 9.2 Hz, Py), 8.20ꢀ8.24 (m, 3H, Py), 8.14 (d, 1H, J = 9.2 Hz, Py),
8.08ꢀ8.10 (d, 1H, J = 9.1 Hz, Py), 7.99ꢀ8.04 (m, 2H, Py), 7.86ꢀ7.89 (m,
2H, H6, Py), 7.43ꢀ7.45 (m, 2H, DMTr), 7.24ꢀ7.36 (m, 7H, DMTr),
6.90ꢀ6.92 (m, 4H, DMTr), 6.25 (d, 1H, J = 3.2 Hz, H10), 5.69 (d, 1H, ex,
J = 6.8 Hz, 30-OH), 5.38 (d, 1H, J = 8.2 Hz, H5), 5.35ꢀ5.37 (m, 1H, H20),
7127
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ARTICLE
solution. The organic phase was washed with satd aq NaHCO3 (10 mL)
and evaporated to near dryness, and the resulting residue was purified by
silica gel column chromatography (40ꢀ70% EtOAc in petroleum ether,
v/v) to afford the corresponding phosphoramidite 4 (yields specified
below).
9H, DMTr), 6.85ꢀ6.89 (m, 4H, DMTr), 6.43 (d, 1H, J = 8.2 Hz, H10),
5.56 (d, 1H, ex, J = 5.2 Hz, 30-OH), 5.43 (dd, 1H, J = 8.3 Hz, 1.7 Hz, H5),
4.41ꢀ4.50 (m, 3H, CH2Py, H30), 4.06ꢀ4.08 (m, 1H, H40), 3.71 (s, 3H,
CH3O), 3.70 (s, 3H, CH3O), 3.44ꢀ3.48 (dd, 1H, J = 8.3 Hz, 8.1 Hz,
H20), 3.28ꢀ3.31 (m, 1H, H50, overlap with H2O), 3.16ꢀ3.20 (dd, 1H,
J = 10.6 Hz, 3.6 Hz, H50), 2.33 (s, 3H, CH3N); 13C NMR (DMSO-d6) δ
162.7, 158.08, 158.07, 150.6, 144.5, 140.2 (C6), 135.4, 135.1, 132.7,
130.7, 130.3, 130.2, 129.71 (DMTr), 129.67 (DMTr), 129.2, 128.0 (Py),
127.8 (DMTr), 127.6 (DMTr), 127.3 (Py), 126.9 (Py), 126.8 (Py),
126.7 (DMTr), 126.1 (Py), 125.01, 124.98 (Py), 124.4 (Py), 124.1,
124.0 (Py), 123.9, 113.20 (DMTr), 113.17 (DMTr), 102.1 (C5), 85.9,
85.1 (C40), 83.4 (C10), 71.3 (C30), 67.8 (C20), 64.1 (C50), 57.4
(CH2Py), 55.0 (CH3O), 38.6 (CH3N). 13C NMR data recorded in
CDCl3 are in agreement with literature reports where 6Q was obtained
via a different synthetic route.15 Full experimental details on the prepara-
tion and characterization of 6Q have not been previously published.
20-Amino-20-deoxy-20-N-methyl-20-N-(pyren-1-yl-carbonyl)-
50-O-(4,40-dimethoxytrityl)uridine (6S). Nucleoside 527 (150 mg,
0.27 mmol) was coevaporated with anhydrous 1,2-dichloroethane (2 ꢁ
5 mL), dissolved in anhydrous DMF (4.5 mL), and added to a prestirred
(1 h at rt) solution of 1-pyrenecarboxylic acid (100 mg, 0.40 mmol),
O-(7-azabenzotriazole-1-yl)-N,N,N0,N0-tetramethyluronium hexafluor-
ophosphate (HATU, 125 mg, 0.32 mmol) and DIPEA (0.12 mL,
0.70 mmol) in anhydrous DMF (4.5 mL). The reaction mixture was
stirred for 17 h, whereupon it was diluted with EtOAc (50 mL) and
sequentially washed with satd aq NaHCO3 (20 mL) and H2O (2 ꢁ
20 mL). The organic phase was evaporated to dryness, and the resulting
residue was purified by silica gel column chromatography (0ꢀ2% MeOH
in CH2Cl2, v/v) to afford nucleoside 6S as a white foam (164 mg, 78%).
Rf 0.4 (5% MeOH in CH2Cl2, v/v). MALDI-HRMS m/z 788.2985
20-O-(Napth-2-yl)-30-O-(N,N-diisopropylamino-2-cyanoetho-
xyphosphinyl)-50-O-(4,40-dimethoxytrityl)uridine (4W). Nu-
cleoside 3W (100 mg, 0.15 mmol), DIPEA (106 μL, 0.59 mmol), and
PCl reagent (66 μL, 0.23 mmol) in anhydrous CH2Cl2 (1.5 mL) were
mixed, reacted, worked up, and purified as described above to afford
phosphoramidite 4W (95 mg, 74%) as a white foam. Rf 0.8 (5% MeOH
in CH2Cl2, v/v); MALDI-HRMS m/z 895.3462 ([M + Na]+, C49H53-
N4O9P Na+, calcd 895.3448); 31P NMR (CDCl3) δ 151.0, 150.9. The
3
reaction yield compares favorably with an independently developed
protocol utilizing 2-cyanoethyl N,N,N0,N0-tetraisopropylphosphorodia-
midite (PN2 reagent) and 1H-tetrazole as phosphitylation reagent
and activator, respectively (∼61% yield).40 The 31P NMR data are in
agreement with literature data.43
30-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-
20-O-(pyren-1-yl)-50-O-(4,40-dimethoxytrityl)uridine (4X). Nucleo-
side 3X (0.28 g, 0.38 mmol), DIPEA (268 μL, 1.50 mmol), and PCl
reagent (167 μL, 0.75 mmol) in anhydrous CH2Cl2 (2.5 mL) were mixed,
reacted, worked up, and purified as described above to afford phosphor-
amidite 4X (0.27 g, 76%) as a white foam. Rf 0.8 (5% MeOH in CH2Cl2,
v/v); MALDI-HRMS m/z 969.3608 ([M + Na]+, C55H55N4O9P Na+,
3
calcd 969.3604); 31P NMR (CDCl3) δ 149.8, 149.4.
30-O-(N,N-Diisopropylamino-2-cyanoethoxyphosphinyl)-
20-O-(pyren-1-yl-methyl)-50-O-(4,40-dimethoxytrityl)uridine
(4Y). Nucleoside 3Y (0.58 g, 0.76 mmol), DIPEA (0.53 mL, 3.05 mmol),
and PCl reagent (340 μL, 1.53 mmol) in anhydrous CH2Cl2 (5 mL)
were mixed, reacted, worked up, and purified as described above to
afford phosphoramidite 4Y (0.56 g, 76%) as a white foam. Rf 0.8 (5%
MeOH in CH2Cl2, v/v); MALDI-HRMS m/z 983.3767 ([M + Na]+,
1
([M + H]+, C48H41N3O8 H+, calcd 788.2966); H NMR (CDCl3)
3
δ 8.65 (br s, 1H, NH, ex), 7.97ꢀ8.28 (m, 9H, Py), 7.85 (d, 1H, J =
8.3 Hz, H6), 7.20ꢀ7.44 (m, 9H, DMTr), 6.80ꢀ6.90 (m, 4H, DMTr),
6.78 (d, 1H, J = 6.0 Hz, H10), 5.42ꢀ5.48 (m, 1H, H5), 4.80ꢀ4.90 (m,
2H, H20, H30), 4.28ꢀ4.43 (m, 1H, H40), 3.77 (s, 6H, CH3O), 3.48ꢀ3.63
(m, 2H, H50), 2.98 (s, 3H, NCH3), traces of a second rotamer are
observed; 13C NMR (CDCl3) δ 174.4, 162.9, 159.0, 150.5, 144.5, 140.0
(C6), 135.6, 135.5, 132.3, 131.4, 131.0, 130.44 (DMTr), 130.42 (DMTr),
129.4(Py), 128.7(Py), 128.5(DMTr), 128.3 (DMTr), 127.4, 126.7 (Py),
126.1 (Py), 126.0 (Py), 125.0 (Py), 124.8, 124.7, 124.3 (Py), 113.6
(DMTr), 113.3 (DMTr), 103.2 (C5), 87.3, 86.6 (C40), 85.3 (C10), 71.8
(C30/C20), 65.8 (C20/C30), 63.0 (C50), 55.5 (CH3O), 38.5 (NCH3).
A trace impurity of grease was observed at 29.9 ppm.
C56H57N4O9P Na+, calcd 983.3761); 31P NMR (CDCl3) δ 150.3,
3
150.2. The observed reaction yield and 31P NMR data are comparable
to the reported protocol that utilizes PN2 reagent and 1H-tetrazole as
phosphitylation reagent and activator, respectively.14 HRMS data have
not been previously reported for this compound.
20-O-(Coronen-1-yl-methyl)-30-O-(N,N-diisopropylamino-
2-cyanoethoxyphosphinyl)-50-O-(4,40-dimethoxytrityl)-
uridine (4Z). Nucleoside 3Z (240 mg, 0.28 mmol), DIPEA (200 μL,
1.11 mmol), and PCl reagent (125 μL, 0.56 mmol) in anhydrous
CH2Cl2 (6 mL) were mixed, reacted, worked up, and purified as des-
cribed above to afford phosphoramidite 4Z (230 mg, 78%) as a light
yellow foam. Rf 0.8 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS m/z
20-Amino-20-deoxy-20-N-methyl-20-N-(pyren-1-yl-methyl-
carbonyl)-50-O-(4,40-dimethoxytrityl)uridine (6V). Known nu-
cleoside 527 (158 mg, 0.28 mmol) was coevaporated with 1,2-dichlor-
oethane (2 ꢁ 5 mL) and subsequently dissolved in anhydrous CH2Cl2
(8 mL). To this was added 1-ethyl-3-(3-dimethylamino-propyl) carbo-
1081.3864 ([M + Na]+, C64H59N4O9P Na+, calcd 1081.3917); 31P
3
NMR (CDCl3) δ 150.2.
20-Amino-20-deoxy-20-N-methyl-20-N-(pyren-1-yl-methyl)-
50-O-(4,40-dimethoxytrityl)uridine (6Q). Known nucleoside
527 (200 mg, 0.36 mmol) was coevaporated with anhydrous 1,
2-dichloroethane (2 ꢁ 4 mL) and redissolved in anhydrous THF
(5 mL). Pyrene-1-ylmethylchloride (205 mg, 0.37 mmol) and triethy-
lamine (0.52 mL, 3.73 mmol) were added, and the reaction mixture was
heated at reflux for 2 days, whereupon the solvent was evaporated off.
The crude residue was taken up in a mixture of CHCl3 and satd aq
NaHCO3 (50 mL, 3:2, v/v), and the layers were separated. The aqueous
phase was extracted with CHCl3 (2 ꢁ 20 mL), and the combined
organic phase was evaporated to dryness. The resulting residue was
purified by silica gel column chromatography (0ꢀ1.25% MeOH/
CH2Cl2, v/v) to afford nucleoside 6Q as a yellow foam (129 mg,
46%). Rf 0.5 (60%, EtOAc in petroleum ether, v/v); MALDI-HRMS m/
diimide hydrochloride (EDC HCl, 73 mg, 0.38 mmol) and pyrene-1-
3
ylacetic acid (108 mg, 0.41 mmol). The reaction mixture was stirred
under argon at rt for 3 h whereupon the reaction mixture was diluted
with CH2Cl2 (30 mL) and sequentially washed with satd aq NaHCO3
(20 mL) and H2O (3 ꢁ 15 mL). The organic phase was evaporated to
dryness and the resulting residue was purified by silica gel column
chromatography (0ꢀ3% i-PrOH/CH2Cl2, v/v) to afford a rotameric
mixture (2:5 by 1HNMR) of nucleoside 6V (187 mg, 83%) as a brown
foam. Rf 0.5 (5%, i-PrOH in CH2Cl2, v/v); MALDI-HRMS m/z
1
801.3078 ([M]+, C49H43N3O8, calcd 801.3045); H NMR (DMSO-
d6) δ 11.52 (d, 1H, J = 1.7 Hz, NH(A)), 11.46 (d, 0.4H, J = 1.7 Hz,
NH(B)), 8.05ꢀ8.31 (m, 11.2H, Py(A) + Py(B)), 7.89 (d, 1H, J = 7.8 Hz,
Py(A)), 7.82 (d, 0.4H, J = 7.8 Hz, Py(B)), 7.67ꢀ7.70 (m, 1.4H, H6(A)
+
1
z 774.3156 ([M + H]+ C48H43N3O7 H+, Calc 774.3174); H NMR
H6(B)), 7.13ꢀ7.43 (m, 12.6H, DMT(A+B)), 6.78ꢀ6.87 (m, 5.6H, DMT-
(A+B)), 6.42 (d, 1H, J = 8.0 Hz, H10(A)), 6.31 (d, 0.4H, J = 5.5 Hz, H10(B)),
5.92 (d, 0.4H, ex, J = 4.9 Hz, 30-OH(B)), 5.76 (d, 1H, ex, J = 4.9 Hz, 30-
3
(DMSO-d6) δ 11.41 (d, 1H, ex, J = 1.7 Hz, NH), 8.50 (d, 1H, J = 9.1 Hz,
Py), 8.01ꢀ8.29 (m, 8H, Py), 7.63 (d, 1H, J = 8.2 Hz, H6), 7.20ꢀ7.38 (m,
7128
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ARTICLE
OH(A)), 5.38 (dd, 1H, J = 8.2 Hz, 1.7 Hz, H5(A)), 5.33 (dd, 0.4H, J =
8.2 Hz, 1.7 Hz, H5(B)), 5.11ꢀ5.17 (m, 1H, H20(A)), 4.80ꢀ4.86 (m,
0.4H, H20(B)), 4.58ꢀ4.63 (d, 1H, J = 16.5 Hz, CH2Py(A)), 4.51ꢀ4.56 (d,
afford phosphoramidite 7V as a bright yellow foam (153 mg, 42%).
Rf 0.4 (60% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z
1024.4037 ([M + Na]+, C58H60N5O9P Na+, calcd 1024.4026); 31PNMR
3
(CDCl3) δ 150.6, 150.5.
0.4H, J = 16.5 Hz, CH2Py(B)), 4.37ꢀ4.49 (m, 2.4H, 1 x CH2Py(A)
,
H30(A), H30(B)), 4.30ꢀ4.37 (d, 0.4H, J = 16.5 Hz, CH2Py(B)), 4.10ꢀ4.16
(m, 1.4H, H40(A) + H40(B)), 3.68 (s, 2.4H, CH3O(B)), 3.64 (s, 3H,
CH3O(A)), 3.62 (s, 3H, CH3O(A)), 3.35ꢀ3.37 (m, 0.4H, H50(B)), 3.32
(s, 3H, CH3N(A)), 3.29ꢀ3.32 (m, 1H, H50(A), overlap with H2O),
3.23ꢀ3.27 (dd, 0.4H, J = 10.6 Hz, 2.6 Hz, H50(B)), 3.15ꢀ3.19 (dd, 1H,
J = 10.6 Hz, 2.6 Hz, H50(A)), 3.03 (s, 1.2H, CH3N(B)); 13C NMR
(DMSO-d6) δ 172.2, 172.1, 162.81, 162.78, 158.06, 158.03, 158.02,
150.5, 150.4, 144.6, 144.3, 140.2 (C6(B)), 140.1 (C6(A)), 135.4, 135.3,
135.2, 135.0, 130.7, 130.62, 130.56, 130.3, 130.2, 129.71 (DMTr),
129.69 (DMTr), 129.1, 128.1 (Py-CHA), 127.9 (Py-CHB), 127.8
(DMTr), 127.73 (DMTr), 127.70 (DMTr), 127.3 (Py), 127.14 (Py),
127.10 (Py), 126.73 (Py), 126.66 (DMTr), 126.1 (Py), 125.1 (Py), 125.0
(Py), 124.9 (Py), 124.5 (Py), 124.1, 124.0, 123.85 (Py), 123.76 (Py), 113.2
(DMTr), 113.1 (DMTr), 102.2 (C5(A)), 102.0 (C5(B)), 85.9, 85.7, 85.5
(C10(B)), 85.2 (C40(A)), 84.8 (C40(B)), 83.2 (C10(A)), 70.5 (C30(A)), 69.3
(C30(B)), 63.8 (C50(A)), 63.7 (C50(B)), 62.1 (C20(B)), 59.0 (C20(A)),
54.92 (CH3O(B)), 54.87 (CH3O(B)), 54.85 (CH3O(A)), 54.82
(CH3O(A)), 38.1 (CH2Py(A)), 37.8 (CH2-Py(B)), 34.2 (CH3N(A)), 31.4
(CH3N(B)).
Synthesis and Purification of ONs. Synthesis of modified
oligodeoxyribonucleotides (ONs) was performed on an DNA synthe-
sizer using 0.2 μmol scale succinyl linked LCAA-CPG (long chain alkyl
amine controlled pore glass) columns with a pore size of 500 Å. Standard
protocols for incorporation of DNA phosphoramidites were used. A
∼50-fold molar excess of modified phosphoramidites in anhydrous
acetonitrile (at 0.05 M) was used during hand-couplings (performed to
conserve material) except with 4Z (∼70-fold molar excess in anhydrous
CH2Cl2, at 0.07M). Moreover, extended oxidation (45 s) and coupling
times were used (0.01 M 4,5-dicyanoimidazole as activator, 15 min for
monomers V, W, and Y, 35 min for monomer Z; 0.01 M 5-(bis-3,5-
trifluoromethylphenyl)-1H-tetrazole [Activator 42], 15 min for mono-
mers Q, S, and V). Cleavage from solid support and removal of
protecting groups were accomplished upon treatment with 32% aq
ammonia (55 °C, 24 h). Purification of all modified ONs was performed
to minimum 80% purity using either of two methods: (a) overall
synthesis yield >80%: cleavage of DMT using 80% aq. AcOH, followed
by precipitation from acetone (ꢀ18 °C for 12ꢀ16 h) and washing with
acetone, or (b) overall synthesis yield <80%: purification of ONs by RP-
HPLC as described below, followed by detritylation and precipitation as
outlined under “a”.
Purification of the crude ONs was performed on a HPLC system
equipped with an XTerra MS C18 precolumn (10 μm, 7.8 ꢁ 10 mm)
and a XTerra MS C18 column (10 μm, 7.8 ꢁ 150 mm) using the
representative gradient protocol depicted in Table S1 in Supporting
Information. The identity of synthesized ONs was established through
MALDI-MS/MS analysis (Tables S2ꢀS3 in Supporting Information)
recorded in positive ions mode on a quadrupole time-of-flight tandem
mass spectrometer equipped with a MALDI source using anthranilic
acid as a matrix, while purity (>80%) was verified by RP-HPLC running
in analytical mode.
Thermal Denaturation Studies. Concentrations of ONs were
estimated using the following extinction coefficients for DNA
(OD/μmol): G (12.01), A (15.20), T (8.40), C (7.05); for RNA
(OD/μmol): G (13.70), A (15.40), U (10.00), C (9.00); for fluoro-
phores(OD/μmol):naphthalene(3.75),18 pyrene(22.4),10 andcoronene
(36.0).44 Each strand was thoroughly mixed and denatured by heating to
70ꢀ85 °C followed by cooling to the starting temperature of the
experiment. Quartz optical cells with a path length of 1.0 cm were
used. Thermal denaturation temperatures (Tm values [°C]) of duplexes
(1.0 μM final concentration of each strand) were measured on a UVꢀvis
spectrophotometer equipped with a 12-cell Peltier temperature con-
troller 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 Na2HPO4 and 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 1 °C). A temperature ramp of 0.5 °C/min
was used in all experiments. Reported Tm values are averages of two
experiments within (1.0 °C.
Steady-State Fluorescence Emission Spectra. Steady-state
fluorescence emission spectra of ONs modified with pyrene-function-
alized monomers Q, S, V, X, and Y and the corresponding duplexes with
cDNA/RNA targets were recorded in nondeoxygenated thermal dena-
turation buffer (each strand 1.0 μM) and obtained as an average of five
scans using an excitation wavelength of λex = 350 nm, excitation slit
5.0 nm, emission slit 2.5 nm, and a scan speed of 600 nm/min.
Experiments were determined at 5 °C to ascertain maximal hybridization
of probes to DNA/RNA targets. Solutions were heated to 80 °C
followed by cooling to 5 °C over 10 min.
20-Amino-20-deoxy-20-N-methyl-20-N-(pyren-1-yl-methyl)-
30-O-(N,N-diisopropylamino-2-cyanoethoxyphosphinyl)-50-
O-(4,40-dimethoxytrityl)uridine (7Q). Nucleoside 6Q (135 mg,
0.18 mmol) was coevaporated with CH3CN (2 ꢁ 4 mL) and redissolved
in anhydrous CH3CN (2.5 mL). To this were added DIPEA (153 μL,
0.87 mmol) and PCl reagent (78 μL, 0.35 mmol). The reaction mixture
was stirred at rt for 4 h, whereupon it was cooled on an ice bath and abs
EtOH (3 mL) was added. The solvent was evaporated off, and the
resulting residue purified by silica gel column chromatography (0ꢀ40%
EtOAc in petroleum ether, v/v; column built in 0.5% Et3N) to afford
nucleoside 6Q as a white foam (97 mg, 57%). Rf 0.3 (40% EtOAc in
petroleum ether, v/v); MALDI-HRMS m/z 996.4046 ([M + Na]+,
C57H60N5O8P Na+, calcd 996.4077); 31P NMR (CDCl3) δ 151.0, 149.8.
3
The 31P NMR data are in general agreement with literature data.15
20-Amino-20-deoxy-20-N-methyl-30-O-(N,N-diisopropylamino-
2-cyanoethoxyphosphinyl)-20-N-(pyren-1-yl-carbonyl)-50-
O-(4,40-dimethoxytrityl)uridine (7S). Nucleoside 6S (219 mg,
0.28 mmol) was coevaporated with anhydrous 1,2-dicholoroethane
(2 ꢁ 2 mL) and redissolved in anhydrous CH2Cl2 (2 mL). To this was
added DIPEA (58 μL, 0.33 mmol) followed by dropwise addition of
PCl reagent (74 μL, 0.33 mmol). After 2 h of stirring at rt, CH2Cl2
(10 mL) was added, and the reaction mixture stirred for additional
10 min, whereupon the solvent was evaporated under reduced pressure.
The resulting residue was purified by silica gel column chromatography
(1st column: 0ꢀ40% EtOAc in petroleum ether, v/v; second column:
0ꢀ4% MeOH in CH2Cl2, v/v) to afford a rotameric mixture of
phosphoramidite 7S as a white foam (138 mg, 49%). Rf 0.8 (5% MeOH
in CH2Cl2, v/v); MALDI-HRMS m/z 1010.3865 ([M + Na]+,
C57H58N5O9P Na+, calcd 1010.3870); 31P NMR (CDCl3) δ 151.8,
3
151.2, 150.8.
20-Amino-20-deoxy-20-N-methyl-30-O-(N,N-diisopropylamino-
2-cyanoethoxyphosphinyl)-20-N-(pyren-1-yl-methylcar-
bonyl)-50-O-(4,40-dimethoxytrityl)uridine (7V). Nucleoside 6V
(0.30 g, 0.37 mmol) was coevaporated with anhydrous 1,2-dicholor-
oethane (2 ꢁ 3 mL) and redissolved in anhydrous CH2Cl2 (4 mL). To
this was added DIPEA (0.32 mL, 1.84 mmol) followed by dropwise
addition of PCl reagent (0.16 mL, 0.74 mmol). After stirring for 1.5 h
at rt, CH2Cl2 (10 mL) was added, and the reaction mixture was stirred
for additional 10 min, whereupon the solvent was evaporated under
reduced pressure. The resulting residue was purified by silica gel
column chromatography (0ꢀ60% EtOAc in petroleum ether, v/v) to
7129
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The Journal of Organic Chemistry
ARTICLE
’ ASSOCIATED CONTENT
54, 3607–3630. (b) Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu, D.;
Minami, T.; Doi, T.; Imanishi, T. Bioorg. Med. Chem. 2001, 9, 1001–1011.
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Supporting Information. Additional discussion regard-
b
ꢂ
ing formation of cyclic N20,O30-hemiaminal ether (Scheme S1);
representative RP-HPLC protocol (Table S1); MALDI-MS of
ONs (Tables S2 and S3); representative thermal denaturation
curves (Figures S1 and S2); discussion of RNA mismatch data for
B2 series (Table S4 and Figure S3); absorption data (Figure S4
and Tables S5 and S6); and steady-state fluorescence emission
spectra (Figures S5ꢀS9) for modified ONs; additional discus-
sion of fluorescence emission spectra; NMR spectra of com-
pounds 2ꢀ7. This material is available free of charge via the
Internet at http://pubs.acs.org.
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*E-mail: hrdlicka@uidaho.edu.
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§These authors contributed equally to this work.
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P.J.H. appreciates support from Award Number R01 GM-
088697 from the National Institute of General Medical Sciences,
National Institutes of Health; Institute of Translational Health
Sciences (ITHS) (supported by grants UL1 RR025014, KL2
RR025015, and TL1 RR025016 from the NIH National Center
for Research Resources); NIH Grant Number P20 RR016454
from the INBRE Program of the National Center for Research
Resources; Idaho NSF EPSCoR and a Univ. Idaho Research
Office and Research Council Seed Grant. S.K. and B.A.A. enjoy
support from the Student Grant Program at University of Idaho
(U80203 & U80077). P.N. and T.B.J. appreciate support from
the The Danish National Research Foundation. We thank Dr.
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Mass Spectrometry Center) for mass spectrometric analyses.
Input from Todd Pankratz, Joanna Hawryluk, and Johanna Root
during preliminary phases of this study and scholarships from the
National Science Foundation under award number 0648202 and
the Department of Defense ASSURE (Awards to Stimulate and
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