Synthesis and biophysical properties of C5-functionalized LNA (locked nucleic acid)

Article abstract of DOI:10.1021/jo500614a

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNAs oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3'-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3'-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.

Full text of DOI:10.1021/jo500614a

Article
pubs.acs.org/joc
Synthesis and Biophysical Properties of C5-Functionalized LNA
(Locked Nucleic Acid)
Pawan Kumar,†,‡ Michael E. Østergaard,† Bharat Baral,† Brooke A. Anderson,† Dale C. Guenther,†
Mamta Kaura,† Daniel J. Raible,† Pawan K. Sharma,‡ and Patrick J. Hrdlicka*,†
†Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343, United States
‡Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
S
* Supporting Information
ABSTRACT: Oligonucleotides modified with conformation-
ally restricted nucleotides such as locked nucleic acid (LNA)
monomers are used extensively in molecular biology and
medicinal chemistry to modulate gene expression at the RNA
level. Major efforts have been devoted to the design of LNA
derivatives that induce even higher binding affinity and
specificity, greater enzymatic stability, and more desirable
pharmacokinetic profiles. Most of this work has focused on
modifications of LNA’s oxymethylene bridge. Here, we
describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases.
Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into
oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and
fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target
affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with
monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease
resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets
with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of
building blocks for RNA-targeting applications and nucleic acid diagnostics.
Many analogues of LNA have been synthesized with the aim
of further improving the binding affinity/specificity, enzymatic
stability and pharmokinetic characteristics of LNA.1,2,16 The
vast majority of these efforts have focused on modifying the
oxymethylene bridge spanning the C2′/C4′-positions and/or
introducing minor-groove-oriented substituents on the bridge.
These structural perturbations have resulted in improved
enzymatic stability and altered biodistribution and/or toxicity
profiles but have generally not resulted in major improvements
in binding affinity and specificity.
C5-functionalized pyrimidine DNA monomers have also
attracted considerable attention,17,18 as they enable predictable
positioning of functional entities in the major groove of nucleic
acid duplexes.19 Small C5-alkynyl substituents such as propyn-
1-yl and 3-aminopropyn-1-yl induce considerable duplex
thermostabilization relative to unmodified duplexes, while
large hydrophobic substituents typically decrease duplex
thermostability. Attachment of polarity-sensitive fluorophores
to the C5 position of DNA pyrimidine monomers has
produced several interesting oligonucleotide probes for
structural studies of nucleic acids and applications in nucleic
acid diagnostics.12c,20
INTRODUCTION
■
The development of novel conformationally restricted nucleo-
tides is a vibrant area of research.1,2 Efforts are driven by the
interesting properties of oligodeoxyribonucleotides (ONs)
modified with classic examples of conformationally restricted
nucleotides such as homo-DNA,3 hexitol nucleic acid (HNA),4
cyclohexane nucleic acid (CeNA),5 bicyclo DNA,6 tricyclo
DNA,7 or locked nucleic acid (LNA),8,9 which is also known as
bridged nucleic acid (BNA).10 ONs comprising these building
blocks display high affinity toward complementary DNA/RNA
often due to reduced entropic binding penalties11 and are
accordingly in high demand for a wide range of nucleic acid
targeting applications in molecular biology, biotechnology, and
pharmaceutical science.12 Their use as RNA-targeting antisense
oligonucleotides to decrease gene expression is a particularly
prominent example.12b
LNA is an especially interesting member of this compound
class because it induces some of the greatest duplex
stabilizations observed to date (Figure 1).8−10 Modulation of
gene expression through LNA-mediated targeting of mRNA,
pre-mRNA, or miRNA has accelerated gene function studies
and led to the development of LNA-based drug candidates
against diseases of genetic origin.13,14 Other applications of
LNA include its use as an in situ hybridization probe to
monitor spatiotemporal expression patterns of miRNAs.15
Received: March 15, 2014
Published: May 5, 2014
© 2014 American Chemical Society
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Figure 1. Structure of LNA-T and C5-functionalized analogues thereof studied herein.
a
Scheme 1. Synthesis of C5-Alkynyl-Functionalized LNA Uridine Phosphoramidites
a
Abbreviations: CAN, ceric ammonium nitrate; DMTrCl, 4,4′-dimethoxytrityl chloride; PCl, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite;
DIPEA, N,N′-diisopropylethylamine.
In light of the above and our ongoing interest in LNA
chemistry,12c,21 we recently set out to study C5-alkynyl-
functionalized LNA uridine (U) monomers, on the basis of
the hypothesis that these monomers will exhibit beneficial
properties from both compound classes, i.e., high affinity
toward RNA complements and good mismatch discrimination
(LNA), along with the ability to position blocking groups in the
major groove to confer protection against enzymatic degrada-
tion (C5 substituent). The results from our preliminary studies
have been very encouraging.22 ONs modified with small C5-
alkynyl-functionalized LNA-U monomers display high affinity
toward complementary RNA and moderate protection against
3′-exonucleases, while ONs modified with large C5-alkynyl-
functionalized LNA-U monomers display greatly increased
enzymatic stability but decreased RNA affinity.
In the present article, we describe full experimental details
concerning the synthesis of 12 different C5-functionalized
LNA-U phosphoramidites, their incorporation into ONs, and
the characterization of these modified ONs by means of
thermal denaturation experiments, analysis of thermodynamic
parameters, nuclease stability experiments, and fluorescence
spectroscopy. The monomers in question were selected to
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ensure a representation of substituents with different sizes,
polarities, linker chemistries, and fluorescence characteristics
(Figure 1).
single incorporation in the 5′-GTGABATGC context are
denoted K1−M1 and so on. Similar conventions apply for ONs
in the B2−B4 series (Table 1). Reference DNA and RNA
strands are denoted D1/D2 and R1/R2, respectively.
RESULTS AND DISCUSSION
Thermal Denaturation Experiments: Binding Affinity.
The thermostabilities of duplexes between modified ONs and
complementary DNA/RNA were evaluated by determining
their thermal denaturation temperature (Tm) in medium salt
phosphate buffer ([Na+] = 110 mM, pH 7.0). Tm’s of modified
duplexes are discussed relative to Tm’s of unmodified reference
duplexes (Table 1).
As anticipated, ONs modified with one or two conventional
LNA-T monomers form very thermostable duplexes with RNA
targets in particular (see ΔTm values for L1−L4, Table 1).
Interestingly, ONs modified with LNA monomers featuring
small C5-alkynyl moieties generally result in the formation of
even more thermostable duplexes (compare ΔTm values of K/
M/N series with those of the L series, Table 1). The effect is
most pronounced for ONs modified with aminopropynyl-
functionalized monomer N, which display increases in Tm
values of up to +13 °C per modification. The greater
thermostability of duplexes modified with K/M/N monomers
is most likely the result of enhanced stacking interactions18a
and, in the case of monomer N, favorable electrostatic
interactions and/or hydration in the major groove, in a manner
similar to that previously suggested for C5-aminopropynyl-
modified DNA.18e,h
In contrast, duplexes modified with LNA monomers that are
conjugated to medium-sized hydrophobic C5-alkynyl substitu-
ents are less thermostable than the corresponding LNA-
modified duplexes (compare ΔTm values of the O/P-series with
those of the L series, Table 1). The trend is particularly
prominent in DNA:RNA duplexes, presumably due to a
suboptimal fit of the C5-alkynyl substituent in the narrow
major groove of A/B-type duplexes. However, other factors,
such as different influences on hydration,18c cannot be ruled
out. The resulting duplexes are, nevertheless, still significantly
more stable that the unmodified reference duplexes.
■
Synthesis of Phosphoramidites. Our route to target
phosphoramidites 5b−l initiates from LNA uridine diol 1,
which is obtained from commercially available diacetone-α-D-
allose in ∼52% yield (Scheme 1).23 C5-iodination of 1 was
accomplished through treatment with iodine and ceric
ammonium nitrate (CAN) in acetic acid at 80 °C for ∼40
min to afford nucleoside 2 in 87% yield. Prolonged heating
and/or higher reaction temperatures result in the formation of
nonpolar impurities, which complicate purification and reduce
product yield. Subsequent O5′-dimethoxytritylation using
standard conditions afforded the key intermediate 3 in 84%
yield. Terminal alkynes24 were then coupled with 3 under
typical Sonogashira conditions25 to provide C5-alkynyl-
functionalized LNA uridines 4a−j in 53−87% yield. Careful
deoxygenation is critical to the outcome of these reactions, as
they otherwise do not proceed to completion. Finally, O3′-
phosphitylation using 2-cyanoethyl-N,N′-diisopropylchloro-
phosphoramidite afforded target phosphoramidites 5b−j in
43−83% yield.
In order to obtain C5-triazolyl-functionalized LNA uridine
phosphoramidites 5k and 5l, C5-ethynyl-functionalized LNA
uridine 4b (obtained via standard TBAF-mediated desilylation
of 4a) was reacted with 1-azidopyrene26 or 1-azidomethylpyr-
ene27 in a Cu(I)-catalyzed azide alkyne Huisgen 1,3-dipolar
cycloaddition,28 followed by standard O3′-phosphitylation
(Scheme 2).
Scheme 2. Synthesis of C5-Triazolyl-Functionalized LNA
Uridine Phosphoramidites
ONs modified with LNA monomers that are conjugated to
long hydrophobic C5-alkynyl substituents display even lower
affinity toward their targets (see ΔTm values of the Q/S series,
Table 1). It is particularly noteworthy that duplexes involving
the doubly modified Q4 or S4 do not display transitions above
10 °C. Similar observations have been made with doubly
cholesterol-modified 2′-amino-LNA.29 We hypothesize that
interactions between the hydrophobic groups in single-stranded
Q4 or S4 interfere with duplex formation. The fact that DNA
duplexes with interstrand zipper arrangements of two Q
monomers are rather thermostable supports this hypothesis
(see Table S2, Supporting Information).
Similarly, ONs modified with LNA monomers that are
conjugated to large hydrophobic fluorophores generally form
very thermolabile duplexes, regardless of whether the
fluorophore is attached via an alkynyl or triazoyl linker (see
ΔTm values of the V−Z series, Table 1). The use of monomers
in which the fluorophore is attached to the nucleobase via a
short rigid linker, such as in monomers W−Y, results in
particularly unstable duplexes. Once again, we speculate that
these trends reflect a poor fit of the fluorophore in the major
groove; short rigid linkers between the fluorophore and
nucleobase moieties may prevent the fluorophore from
sampling more suitable conformational space. Interestingly,
with the exception of pyrene- and perylene-functionalized W4
ON Synthesis. Phosphoramidites 5b−l were used in
machine-assisted solid-phase DNA synthesis (0.2 μmol scale)
to incorporate monomers K−Z into ONs. Standard conditions
were used except for extended hand-coupling (generally 15 min
with 4,5-dicyanoimidazole or 5-[3,5-bis(trifluoromethyl)-
phenyl]-1H-tetrazole as activator) when using 5b−l, which
typically resulted in stepwise coupling yields of >95%. The
composition and purity of all modified ONs was ascertained by
MALDI MS analysis (Table S1, Supporting Information) and
ion-pair reversed-phase HPLC, respectively. ONs containing a
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Table 1. ΔTm Values of Duplexes between ONs Modified with C5-Functionalized LNA Monomers and Complementary DNA/
a
RNA Measured Relative to Unmodified Duplexes
ΔTm/mod (°C)
ON
duplex
B =
L
K
M
N
O
P
Q
S
V
W
X
Y
Z
B1
5′-GTG ABA TGC
3′-CAC TAT ACG
+5.0
+7.0
+7.0
+8.0
+4.5
+4.0
+1.0
−5.5
−6.5
−8.5
−12.5
−10.5
−5.5
D2
D1
B2
5′-GTG ATA TGC
3′-CAC BAT ACG
+4.0
+6.5
+5.5
+9.5
+6.5
+9.5
+8.0
+5.5
+5.5
+5.5
+11.0
+8.5
+8.5
+8.5
+5.5
+7.0
+5.5
+9.5
+8.0
+10.0
+8.0
+6.5
+9.5
+3.0
+4.5
nd
+1.0
+3.5
+3.0
+5.5
+2.0
+2.0
+4.5
+0.5
+1.0
−5.0
−3.5
<−10.0
−2.0
0
−7.5
−4.0
+0.5
−4.0
−6.0
0
−9.5
−10.5
−6.5
−2.0
−1.5
−5.5
−5.5
−12.0
−11.5
<−10.0
−12.0
−12.0
−11.0
<−8.5
−13.5
−12.5
−4.0
−6.5
−5.5
−2.0
−1.5
−5.0
−1.0
−0.5
D1
B3
5′-GTG ATA TGC
3′-CAC TAB ACG
D1
B4
5′-GTG ATA TGC
3′-CAC BAB ACG
+8.0
<−10.0
+4.0
B1
R2
5′-GTG ABA TGC
3′-CAC UAU ACG
+13.0
+10.0
+12.5
+11.0
+6.0
−0.5
+2.5
nd
−2.0
R1
B2
5′-GUG AUA UGC
3′-CAC BAT ACG
+3.5
−10.0
−9.0
R1
B3
5′-GUG AUA UGC
3′-CAC TAB ACG
+2.5
−1.0
<−8.5
R1
B4
5′-GUG AUA UGC
3′-CAC BAB ACG
<−8.5
+2.0
−2.0
a
ΔTm = change in Tm values relative to unmodified reference duplexes D1:D2 (Tm  29.5 °C), D1:R2 (Tm  27.0 °C), and D2:R1 (Tm  27.0
°C); Tm values were determined as the first-derivative maximum of denaturation curves (A260 vs T) recorded in medium salt phosphate buffer ([Na+]
= 110 mM, [Cl−] = 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 μM of each strand. Tm values are averages of at least two measurements
within 1.0 °C; see Figure 1 for structures of monomers. nd = not determined. Data for duplexes between L/K/N/Q/S-modified ONs and
complementary RNA has been previously reported in ref 22.
and X4, duplexes entailing the doubly modified B4 ONs are
considerably more stable than those entailing their singly
modified counterparts (e.g., compare ΔTm/mod of B4:D1
relative to B2:D1 and B3:D1, Table 1). Similar stabilizing
trends have been reported for other densely fluorophore
modified duplexes and were attributed to the formation of
chromophore arrays in the major groove.19 The presence of
pyrene excimer signals in the steady-state fluorescence emission
spectra of duplexes between V4/Y4/Z4 and DNA/RNA
complements supports this hypothesis (Figure S3, Supporting
Information).
Thermodynamic Analysis of Duplexes Modified with
C5-Functionalized LNA-U Monomers. The Tm-based
conclusions are largely corroborated through analysis of the
thermodynamic parameters for duplex formation, which were
derived from thermal denaturation curves through curve
fitting.30 Thus, the formation of duplexes between conventional
LNA L1−L3 and complementary DNA or RNA is 4−7 and 8−
13 kJ/mol more favorable, respectively, in comparison to
unmodified reference duplexes (see ΔΔG298 values for L1−L3,
Table 2). The greater stability of LNA-modified duplexes is
generally a result of lower enthalpy (ΔΔH < 0 kJ/mol for L2
and L3, Table 2), but entropic stabilization is also observed
(Δ(T298ΔS) < 0 kJ/mol for L1, Table 2).
which is consistent with improved base stacking due to the
extended π surface of the C5-alkynyl-functionalized LNA
monomers (compare ΔΔH values for the K/M/N series vs L
series, Table 2); similar trends have been previously reported
for C5-propynyl-functionalized DNA monomers.31
Duplexes involving ONs modified with monomers O/P/Q,
which are conjugated to moderately large hydrophobic alkynyl
substituents, are 0−7 kJ/mol less favorable than the
corresponding LNA-modified duplexes (compare ΔΔG298
values for the O/P/Q series vs the L series, Table 2).
Comparison with K-modified duplexes suggests that the
hydrophobic substituents counteract the favorable enthalpy of
the extended π surfaces (compare ΔΔH values for the O/P/Q
series vs the K series, Table 2). One possible interpretation of
this is that the hydrophobic substituents disrupt hydration in
the major groove.
DNA duplexes modified with C5-cholesterol-functionalized
LNA monomer S are less stable than the control duplex,
whereas duplexes with RNA are slightly more stable (see
ΔΔG298 values for S1−S3, Table 2). The favorable enthalpic
contribution of the alkyne functionality is fully counteracted by
low entropy in DNA duplexes but only partially counteracted in
DNA:RNA duplexes (compare ΔΔH vs Δ(T298ΔS) for S1−S3,
Table 2).
Formation of duplexes entailing ONs modified with K/M/N
monomers, which are conjugated to small and/or relatively
polar alkynes, is 1−7 kJ/mol more favorable than formation of
the corresponding LNA-modified duplexes (compare ΔΔG298
values for the K/M/N series vs L series, Table 2). The
additional duplex stabilization is generally enthalpic in origin,
Thermal Denaturation Studies: Binding Specificity.
The binding specificities of centrally modified ONs (B1 series)
were determined by using DNA/RNA targets with mismatched
nucleotides opposite of the modified monomer. As ex-
pected,8−10 LNA-modified ON L1 displays improved binding
specificity relative to unmodified reference strand D1, as
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a
Table 2. Thermodynamic Parameters for Formation of Duplexes Modified with C5-Functionalized LNA Monomers
+complementary DNA
+complementary RNA
ΔG298 [ΔΔG298
]
ΔH [ΔΔH]
−T298ΔS [Δ(T298ΔS)]
(kJ/mol)
ΔG298 [ΔΔG298
]
ΔH [ΔΔH]
−T298ΔS [Δ(T298ΔS)]
ON
sequence
(kJ/mol)
(kJ/mol)
(kJ/mol)
(kJ/mol)
(kJ/mol)
D1
D2
5′-GTG ATA TGC
3′-CAC TAT ACG
−42
−42
−314
−314
271
−36
−39
−278
−293
241
254
271
L1
L2
L3
5′-GTG ALA TGC
3′-CAC LAT ACG
3′-CAC TAL ACG
−47 [−5]
−46 [−4]
−49 [−7]
−297 [+17]
−332 [−18]
−332 [−18]
250 [−21]
286 [+15]
283 [+12]
−49 [−13]
−47 [−8]
−50 [−11]
−309 [−31]
−331 [−38]
−340 [−47]
260 [+19]
283 [+29]
290 [+36]
K1
K2
K3
5′-GTG AKA TGC
3′-CAC KAT ACG
3′-CAC TAK ACG
−49 [−7]
−49 [−7]
−52 [−10]
−350 [−36]
−349 [−35]
−372 [−58]
301 [+30]
300 [+29]
319 [+48]
−53 [−17]
−49 [−10]
−57 [−18]
−424 [−146]
−367 [−74]
−414 [−121]
371 [+130]
317 [+63]
357 [+103]
M1
M2
M3
5′-GTG AMA TGC
3′-CAC MAT ACG
3′-CAC TAM ACG
−51 [−9]
−50 [−8]
−51 [−9]
−390 [−76]
−394 [−80]
−360 [−46]
339 [+68]
344 [+73]
309 [+38]
−52 [−16]
−51 [−12]
−51 [−12]
−386 [−108]
−398 [−105]
−367 [−74]
334 [+93]
347 [+93]
316 [+62]
N1
N2
N3
5′-GTG ANA TGC
3′-CAC NAT ACG
3′-CAC TAN ACG
−51 [−9]
−49 [−7]
−52 [−10]
−353 [−39]
−362 [−48]
−361 [−47]
302 [+31]
313 [+42]
309 [+38]
−51 [−15]
−52 [−13]
−52 [−13]
−324 [−46]
−364 [−71]
−325 [−32]
272 [+31]
312 [+58]
272 [+18]
O1
O2
O3
5′-GTG AOA TGC
3′-CAC OAT ACG
3′-CAC TAO ACG
−47 [−5]
−44 [−2]
−46 [−4]
−337 [−23]
−322 [−8]
−324 [−10]
290 [+19]
278 [+7]
278 [+7]
−46 [−10]
−43 [−4]
−44 [−5]
−337 [−59]
−366 [−73]
−340 [−47]
291 [+50]
322 [+68]
296 [+42]
P1
P2
P3
5′-GTG APA TGC
3′-CAC PAT ACG
3′-CAC TAP ACG
−45 [−3]
−43 [−1]
−44 [−2]
−334 [−20]
−324 [−10]
−339 [−25]
289 [+18]
281 [+10]
294 [+23]
−45 [−9]
−43 [−4]
−43 [−4]
−327 [−49]
−351 [−58]
−365 [−72]
282 [+41]
308 [+54]
321 [+67]
Q1
Q2
Q3
5′-GTG AQA TGC
3′-CAC QAT ACG
3′-CAC TAQ ACG
−45 [−3]
−45 [−3]
−43 [−1]
−346 [−32]
−411 [−97]
−287 [+27]
301 [+30]
371 [+100]
243 [−28]
−45 [−9]
−46 [−7]
−43 [−4]
−347 [−69]
−377 [−84]
−360 [−67]
302 [+61]
331 [+77]
317 [+63]
S1
S2
S3
5′-GTG ASA TGC
3′-CAC SAT ACG
3′-CAC TAS ACG
−37 [+5]
−39 [+3]
−40 [+2]
−317 [−3]
−380 [−66]
−380 [−66]
280 [+9]
342 [+71]
339 [+68]
−39 [−3]
−42 [−3]
−40 [−1]
−333 [−55]
−359 [−66]
−355 [−62]
294 [+53]
316 [+62]
315 [+61]
a
Parameters were determined from thermal denaturation curves, which were recorded as described in Table 1. ΔΔG298, ΔΔH, and Δ(T298ΔS) are
calculated relative to reference duplexes D1:D2, D1:R2, and D2:R1.
evidenced by the more pronounced decreases in Tm values of
mismatched duplexes (compare ΔTm values for L1 and D1,
Table 3). Interestingly, many of the C5-functionalized LNA
monomers induce additional improvements in binding
specificity (note ΔTm values of K1/M1/N1/O1/P1/Q1,
Table 3). It is recognized that nucleotide modifications,
which improve target affinity as well as binding specificity, are
desirable for nucleic acid targeting applications.32 Cholesterol-
functionalized LNA S1 and fluorophore-functionalized LNAs
V1/W1/X1/Y1/Z1 display poor discrimination of mismatched
DNA targets but maintain reasonable specificity against RNA
targets (Table 3). These trends are indicative of different
binding modes of the pyrene and perylene moieties in
DNA:DNA vs DNA:RNA duplexes. Intercalation of aromatic
units, which is known to stabilize mismatched base pairs,33 is
more favorable in DNA:DNA than in DNA:RNA duplexes.34
For a discussion of the binding specificities of double-modified
ONs (B4-series), see the Supporting Information (Table S3).
3′-Exonuclease Stability of C5-Functionalized LNA.
Next, we examined the enzymatic stability of select C5-
functionalized LNAs and reference strands in the presence of
snake venom phosphodiesterase (SVPDE), a 3′-exonuclease. As
expected, unmodified D2 is quickly degraded (>95% cleavage
after 15 min), while the singly modified LNA L2 offers
moderate protection against SVPDE (>95% cleaved after 50
min) (Figure 2). ONs modified with a single C5-ethynyl- or
C5-aminopropynyl-functionalized LNA monomer are markedly
more resistant toward SVPDE degradation (∼55% and 35%
cleavage of K2 and N2, respectively, after 2 h). Interestingly,
ONs that are modified with LNA monomers conjugated to
large hydrophobic substituents are completely inert against
SVPDE-mediated degradation, following a brief period of
cleavage (see degradation profiles for Q2 and S2; Figure 2).
M2/O2/P2 also display markedly increased 3′-exonuclease
resistance (Figure S1, Supporting Information). As expected,
these trends are even more pronounced with the doubly
modified B4 series (Figure 2). Thus, the data strongly suggest
that large hydrophobic C5-alkynyl substituents offer effective
protection from enzymatic degradation.
Fluorescence Properties of C5-Functionalized LNA.
Steady-state fluorescence emission spectra of ONs modified
with C5-fluorophore-functionalized LNA monomers and the
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a
Table 3. Discrimination of Mismatched DNA/RNA Targets by Singly Modified LNAs and Reference ONs
DNA: 3′-CAC TBT ACG
ΔTm
RNA: 3′-CAC UBU ACG
ΔTm
Tm
Tm
ON
sequence
A
C
G
T
A
C
G
U
D1
L1
5′-GTG ATA TGC
5′-GTG ALA TGC
5′-GTG AKA TGC
5′-GTG AMA TGC
5′-GTG ANA TGC
5′-GTG AOA TGC
5′-GTG APA TGC
5′-GTG AQA TGC
5′-GTG ASA TGC
5′-GTG AVA TGC
5′-GTG AWA TGC
5′-GTG AXA TGC
5′-GTG AYA TGC
5′-GTG AZA TGC
29.5
34.5
36.5
36.5
37.5
34.0
33.5
30.5
24.0
23.0
21.0
17.0
19.0
24.0
−16.5
−18.0
−20.0
−20.0
−19.0
−20.5
−21.5
−18.0
−11.5
−7.5
−8.0
−11.0
−15.5
−11.5
−12.0
−16.5
−17.0
−13.0
−10.0
−10.0
−7.0
−15.5
−16.0
−18.5
−18.5
−17.5
−18.0
−20.5
−16.5
−11.0
−7.5
27.0
36.5
38.0
36.5
40.0
33.0
32.5
31.0
25.0
23.0
25.0
15.0
25.0
25.5
<−17.0
−19.0
−20.5
−18.5
−18.5
−20.0
−20.5
−19.5
−15.0
<−13.0
<−15.0
<−5.0
<−15.0
−1.5
−4.5
−8.0
−13.5
−9.5
−11.5
−9.5
−11.5
−10.0
−9.0
−10.5
<−15.0
<−5.0
−8.0
<−17.0
−18.5
−22.0
−20.0
−22.5
−20.0
−19.5
−20.0
<−15.0
<−13.0
<−15.0
<−5.0
<−15.0
<−15.5
K1
M1
N1
O1
P1
Q1
S1
V1
W1
X1
Y1
Z1
+6.0
+3.0
+4.5
0.0
+3.5
−1.0
−10.0
−4.0
<−14.0
−2.5
−9.5
<−15.5
a
For experimental conditions and sequences, see Table 1. ΔTm = change in Tm value relative to fully matched ON:DNA or ON:RNA duplex (B =
A). Data for L1/K1/N1/Q1/S1 against mismatched RNA have been previously published in ref 22.
Figure 2. 3′-Exonuclease (SVPDE) degradation of singly (left, 3′-CAC BAT ACG) and doubly modified (right, 3′-CAC BAB ACG) C5-
functionalized LNA and reference strands. Nuclease degradation studies were performed in magnesium buffer (50 mM Tris-HCl, 10 mM Mg2+, pH
9.0) by using 3.3 μM ONs and 0.03 U of SVPDE. Data depicted in the left panel have been previously reported in ref 22.
corresponding duplexes with complementary or mismatched
DNA targets were recorded to gain further insight into the
binding modes of the fluorophores. In addition to studying the
fluorescence properties of B1 and B4 probes in the presence or
absence of matched/mismatched DNA/RNA (Figures S2 and
S3, Supporting Information), we also studied centrally modified
13-mer ONs (V5−Z5 series) and their duplexes with matched/
mismatched DNA targets (Figure 3). The thermal denaturation
characteristics of these ONs (Table S4, Supporting Informa-
tion) closely follow those of the singly modified 9-mer ONs: i.e.
(i) the corresponding duplexes with DNA/RNA targets are less
stable than unmodified reference duplexes (only Z5-modified
duplexes are slightly more stable) and (ii) W5−Y5 display very
poor thermal discrimination of mismatched DNA targets, while
V5 and Z5 display similar binding specificity as the unmodified
reference strands.
V/Y/Z-modified duplexes exhibit emission peaks of varying
broadness at ∼390/402 nm (V), ∼381/398 nm (Y), and
∼376/396/416 nm (Z), respectively, which are typical emission
maxima for electronically isolated pyrene units (Figure 3). As
expected for duplexes modified with the 1-ethynylpyrene
fluorophore,35 the duplex between W5 and complementary
DNA exhibits broad red-shifted emission centered around
∼465 nm, which is indicative of strong electronic coupling
between the pyrene and nucleobase moiety. Interestingly, the
emission intensities of pyrene-functionalized ONs V5/W5/Y5/
Z5 increase upon binding to complementary DNA (∼3.8-,
∼3.9-, ∼3.1-, and ∼51-fold increases for V5, W5, Y5 and Z5,
respectively, Figure 3). In contrast, much smaller increases are
observed upon hybridization with mismatched DNA targets.
The intensity differences are most likely due to different
positioning of the pyrene moieties in matched vs mismatched
duplexes, in a manner similar to that proposed for the
corresponding DNA analogues of monomers V/W/Y/Z.18g,j,35b
Thus, the pyrene moieties likely point into the nonquenching
environment of the major groove in matched duplexes
(nucleobase in anti conformation), while they are intercalating
into mismatched duplexes leading to nucleobase-mediated
quenching36 of pyrene fluorescence (nucleobase in syn
conformation). Regardless of the mechanism, the results
strongly suggest that V/W/Y/Z-modified ONs are promising
probes for the detection of nucleic acid targets and fluorescent
discrimination of single-nucleotide polymorphisms (SNPs).
Duplexes between perylene-functionalized X5 and comple-
mentary DNA display broad emission maxima at ∼487 and
∼517 nm, whereas the emission maxima are red-shifted by ∼10
nm in mismatched DNA duplexes (Figure 3). The emission
intensity of X5 does not change significantly upon binding with
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Figure 3. Steady-state fluorescence emission spectra of single-stranded B5 ONs (5′-CG CAA CBC AAC GC) and the corresponding duplexes with
fully complementary or singly mismatched DNA strands (mismatched nucleotide opposite of modification is specified). Conditions: λex 344 nm
(V5/Y5/Z5), λex 375 nm (W5), λex 448 nm (X5); T = 5 °C. Note that different axis scales are used.
complementary DNA but is reduced by 30−60% upon binding
to mismatched targets, presumably due to nucleobase-mediated
quenching of intercalating perylene units.
The large increases in fluorescence intensity upon hybrid-
ization of Z5 with complementary targets prompted us to
examine the potential of Z-modified ONs as hybridization
probes38 in greater detail. Three additional 13-mer ONs were
therefore prepared in which the nucleotides flanking monomer
Z were systematically varied (Table S4, Supporting Informa-
tion). Although the increases in fluorescence intensities upon
hybridization with DNA targets are less pronounced (4−17-
fold, Figure 5) and the resulting duplexes are significantly less
fluorescent than with Z5,39 moderate to excellent fluorescent
discrimination of mismatched DNA targets is observed with all
Z-modified probes (discrimination factors from 1.5 to 48,
Figure 5). Accordingly, Z-modified ONs constitute an
interesting addition to the existing pool of pyrene-based
hybridization probes.18j,40
Recently, we examined the SNP-discriminating properties of
V-modified ONs and compared them to probes modified with
the corresponding DNA analogue of monomer V.37 We found
that there are distinct advantages to conjugating the 1-
pyrenecarboxamido fluorophore to the C5-position of LNA-
U, including (i) greater increases in fluorescence intensity upon
target binding, (ii) formation of more brightly fluorescent
duplexes, and (iii) stricter fluorescent discrimination of DNA
targets with SNP sites. Force field calculations suggested that
the extreme pucker of the LNA skeleton influences the
rotational freedom around the N1−C1′ glycosyl bond due to
steric hindrance between H6 and H3′, leading to different
positioning and modulated photophysical properties of the C5-
fluorophore relative to the analogous DNA monomer.37
Direct comparison of Y5 and Z5 with the corresponding
DNA-based probes Y5d and Z5d18j (for structures of the DNA
analogues of V/Y/Z monomers, see Figure S4 in the
Supporting Information) reveals similar advantages (Figure
4). Thus, the results suggest that conjugation of fluorophores to
the C5 position of LNA monomers is an effective strategy
toward the generation of building blocks with interesting
photophysical properties.
CONCLUSION
■
The hybridization characteristics and enzymatic stabilities of
ONs modified with LNA uridines can be extensively modulated
through conjugation of different entities to the C5 position of
the nucleobase. Only two extra steps, relative to conventional
LNA synthesis, are needed. Monomers that are conjugated to
small alkynyl substituents result in significantly greater target
affinity and specificity than regular LNA monomers. Con-
jugation of bulky moieties confers complete protection against
3′-exonucleases but also decreases target affinity. ONs modified
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although concerns regarding the potential toxicity of C5-alkynyl
entities41 must be alleviated prior to biological evaluation.
The present study suggests that it is possible to combine
desirable properties from LNA (target affinity/specificity) and
C5-functionalized DNA monomers (positioning of functional
entities in the major groove) into one compound class. The
subsequent article in this issue demonstrates that the properties
of ONs modified with α-L-LNA uridines also can be modulated
through functionalization of the nucleobase.42 We therefore
anticipate that C5 functionalization of pyrimidines will serve as
a general and synthetically straightforward approach for
modulation of pharmocodynamic and pharmacokinetic proper-
ties of oligonucleotides modified with LNA8−10,13 or other
conformationally restricted monomers.1−7,16 Efforts aiming at
delineating whether the biophysical properties of LNA purines
also can be improved through functionalization of the
nucleobase are ongoing, and the results from these studies
will be reported shortly.
EXPERIMENTAL SECTION
■
Representative Protocol for EDC-Mediated Coupling of
Carboxylic Acids with Propargylamine to Furnish Alkynes
Ae−Ag43 Used in Sonogashira Couplings. The appropriate
carboxylic acid and 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide
hydrochloride (EDC·HCl) were added to propargylamine in
anhydrous CH2Cl2, and the reaction mixture was stirred under an
argon atmosphere until analytical TLC indicated full conversion
(quantities, volumes, reaction time, and temperature are specified
below). At this point, CH2Cl2 (40 mL) was added and the organic
phase was washed with 5% aqueous citric acid (2 × 20 mL) and H2O
(20 mL). The aqueous phase was back-extracted with CH2Cl2 (25
mL), the combined organic layers were concentrated to dryness, and
the resulting residue was purified by column chromatography (0−4%
MeOH in CH2Cl2) to afford the desired product (quantities and yields
specified below).
Figure 4. Fluorescence intensity of single-stranded probes (SSPs) in
the presence or absence of complementary or singly mismatched
DNA/RNA strands. Mismatched nucleotide opposite of modification
is specified. Hybridization-induced increases and discrimination factors
(defined as the fluorescence intensity of duplexes with complementary
DNA/RNA divided by the intensity of SSPs or duplexes with
mismatched DNA/RNA, respectively) are given above the corre-
sponding histograms. Intensity recorded at λem 382 nm for Y5/Y5d
and λem 377 nm for Z5/Z5d at T = 5 °C. Note that different y-axis
scales are used.
N-(Prop-2-ynyl)-2-(adamant-1-yl)ethanamide (Ae). 1-Adamanta-
neacetic acid (1.60 g, 8.24 mmol), EDC·HCl (1.80 g, 9.42 mmol), and
propargylamine (0.60 mL, 9.38 mmol) in anhydrous CH2Cl2 (30 mL)
were set up, reacted (14 h at room temperature), and worked up, and
the product was purified as described above to afford alkyne Ae43
(1.40 g, 76%) as a white solid material: Rf = 0.8 (10% MeOH/CH2Cl2,
v/v); MALDI-HRMS m/z 254.1527 ([M + Na]+, C15H21NO·Na+,
1
calcd 254.1515); H NMR (CDCl3) δ 5.56 (br s, 1H, NH), 4.01 (dd,
2H, J = 5.2 Hz, 2.5 Hz, CH2NH), 2.19 (t, 1H, J = 2.5 Hz, HCC),
1.93−1.97 (m, 3H, 3 × CH), 1.92 (s, 2H, CH2CONH), 1.58−1.70
(12H, 6 × CH2-ada); 13C NMR (CDCl3) δ 170.8, 80.0, 71.6 (HC
C), 51.6 (CH2CONH), 42.8 (CH2-ada), 37.0 (CH2-ada), 33.1, 29.2
(CH2NH), 28.9 (CH-ada).
N-(Prop-2-ynyl)dodecanamide (Af). Lauric acid (dodecanoic acid,
1.60 g, 8.00 mmol), EDC·HCl (1.80 g, 9.42 mmol), and propargyl-
amine (0.60 mL, 9.38 mmol) in anhydrous CH2Cl2 (30 mL) were set
up, reacted (12 h at room temperature), and worked up, and the
product was purified as described above to afford alkyne Af43 (1.40 g,
76%) as a white solid material: Rf = 0.8; MALDI-HRMS m/z 260.1978
Figure 5. Fluorescence intensity of single-stranded probes (SSPs) in
the presence or absence of complementary or singly mismatched DNA
strands. Mismatched nucleotide opposite of modification is specified.
Hybridization-induced increases and discrimination factors are given
above the corresponding histograms. Target: 5′-CG CAA BZB AAC
GC, where B = C/A/G/T for ON5−8, respectively. Intensity recorded
at λem 377 nm at T = 5 °C.
1
([M + Na]+, C15H27NO·Na+, calcd 260.1985); H NMR (CDCl3) δ
5.66 (bs, ex, 1H, NH), 4.03 (dd, 2H, J = 5.0 Hz, 2.5 Hz, CH2CCH),
2.19 (t, 1H, J = 2.5 Hz, HCC), 2.16 (2d, 2H, J = 7.7 Hz, CH2CO),
1.61 (quintet, 2H, J = 7.7 Hz, CH2CH2CO), 1.20−1.30 (m, 16H, 8 ×
CH2), 0.85 (t, 3H, J = 6.5 Hz, CH3); 13C NMR (CDCl3) δ 172.9, 79.9,
71.7, 36.7 (CH2CO), 32.1 (CH2), 29.8 (CH2), 29.7 (CH2), 29.53
(CH2), 29.47 (CH2), 29.4 (CH2CCH), 25.8 (CH2CH2CO), 22.9,
with C5-fluorophore-functionalized LNA uridines display
improved photophysical characteristics relative to the corre-
sponding DNA-based probes, including greater hybridization-
induced increases in fluorescence intensity, formation of more
brightly fluorescent duplexes, and strict fluorescent discrim-
44
1
14.3 (CH3). H NMR data are in agreement with literature reports.
N-(Prop-2-ynyl)octadecanamide (Ag). Stearic acid (octadecanoic
acid, 1.42 g, 5.00 mmol), EDC·HCl (1.15 g, 6.00 mmol), and
propargylamine (0.40 mL, 6.25 mmol) in anhydrous CH2Cl2 (30 mL)
were set up, reacted (12 h at room temperature), and worked up, and
the product was purified as described above to afford alkyne Ag43
(1.40 g, 87%) as a white solid material: Rf = 0.1 (CH2Cl2); FAB-
ination of single-nucleotide polymorphisms.20 These proper-
b
ties render C5-functionalized LNA as promising building blocks
for RNA-targeting applications and nucleic acid diagnostics,
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HRMS m/z 321.3020 ([M]+, C21H39NO+, calcd 321.3032); H NMR
(CDCl3) δ 5.54 (br s, 1H, ex, NH), 4.03 (dd, 2H, J = 5.5 Hz, 2.5 Hz,
CH2NH), 2.20 (t, 1H, J = 2.5 Hz, HCC), 2.17 (t, 2H, J = 7.5 Hz,
CH2CO), 1.61 (quintet, 2H, J = 7.5 Hz, CH2CH2CO), 1.23−1.27 (m,
28H, 14 × CH2), 0.87 (t, 1H, J = 7.0 Hz, CH3); 13C NMR (CDCl3) δ
172.7, 79.7, 71.5 (HCC), 36.5 (CH2CO), 31.9 (CH2), 29.69 (CH2),
29.684 (CH2), 29.677 (CH2), 29.66 (CH2), 29.65 (CH2), 29.64
(CH2), 29.59 (CH2), 29.46 (CH2), 29.35 (CH2), 29.31 (CH2), 29.2
(CH2), 29.1 (CH2NH), 25.5 (CH2CH2CONH), 22.7 (CH2), 14.1
(CH3). The NMR data are in excellent agreement with literature
reports.45
(4a). Nucleoside 3 (0.68 g, 1.00 mmol), Pd(PPh3)4 (120 mg, 0.10
mmol), CuI (40 mg, 0.20 mmol), trimethylsilylacetylene (294 mg,
0.42 mL, 3.00 mmol), and Et3N (0.60 mL, 4.27 mmol) in DMF (10
mL) were reacted as described in the representative Sonogashira
protocol, and the mixture was stirred stirred at room temperature for
12 h. After workup and purification, nucleoside 4a (0.56 g, 85%) was
obtained as a brown solid material. Rf = 0.5 (5% MeOH in CH2Cl2, v/
v); ESI-HRMS m/z 677.2297 ([M + Na]+, C36H38N2O8Si·Na+, calcd
1
677.2290); H NMR (DMSO-d6) δ 11.69 (s, 1H, ex, NH), 7.86 (s,
1H, H6), 7.20−7.45 (m, 9H, Ar), 6.89 (d, 4H, J = 8.5 Hz, Ar), 5.72 (d,
1H, ex, J = 4.5 Hz, 3′−OH), 5.39 (s, 1H, H1′), 4.28 (s, 1H, H2′), 4.08
(d, 1H, J = 4.5 Hz, H3′), 3.75−3.80 (m, 2H, 2 × H5″), 3.73 (s, 6H, 2
× OCH3), 3.40−3.43 (d, 1H, J = 11.0 Hz, H5′), 3.30−3.33 (d, 1H, J =
11.0 Hz, H5′, overlap with H2O signal), −0.04 (s, 9H, Me3Si); 13C
NMR (DMSO-d6) δ 161.5, 158.05, 158.03, 148.9, 144.6, 142.5 (C6),
135.4, 135.2, 129.7 (Ar), 129.5 (Ar), 127.8 (Ar), 127.6 (Ar), 126.6
(Ar), 113.2 (Ar), 97.9, 97.5, 96.9, 87.7, 87.1 (C1′), 85.5, 78.6 (C2′),
71.3 (C5″), 69.5 (C3′), 58.9 (C5′), 55.0 (CH3O), −0.47 (Me3Si).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-3-(5-ethynylura-
cil-1-yl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptane (4b). To a sol-
ution of nucleoside 4a (0.53 g, 0.81 mmol) in THF (20 mL) was
added tetrabutylammonium fluoride in THF (TBAF, 1 M, 1.2 mL, 1.2
mmol), and the reaction mixture was stirred at room temperature for 2
h. EtOAc (50 mL) was added and the organic phase washed with brine
(2 × 30 mL) and H2O (30 mL). The aqueous phase was back-
extracted with EtOAc (30 mL). The combined organic phase was
dried (Na2SO4) and evaporated to dryness and the resulting residue
purified by column chromatography (0−5% MeOH in CH2Cl2, v/v)
to afford nucleoside 4b (0.37 g, 78%) as a lightly brown solid material:
Rf = 0.4 (5% MeOH/CH2Cl2, v/v); ESI-HRMS m/z 621.1666 ([M +
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(5-iodoracil-1-
yl)-2,5-dioxabicyclo[2.2.1]heptane (2). To a solution of nucleoside
123 (4.00 g, 15.62 mmol) in glacial AcOH (150 mL) were added
iodine (2.40 g, 9.44 mmol) and ceric ammonium nitrate (4.26 g, 7.77
mmol), and the reaction mixture was stirred at 80 °C for ∼40 min.
After it was cooled to room temperature, the mixture was evaporated
to dryness and the resulting residue was suspended in MeOH (150
mL). The mixture was concentrated and the resulting residue adsorbed
on silica gel and purified by column chromatography (0−15% MeOH/
CH2Cl2, v/v) to afford nucleoside 2 (5.21 g, 87%) as a white solid
material: Rf = 0.4 (10% MeOH/CH2Cl2, v/v); FAB-HRMS m/z
1
382.9732 ([M + H]+, C10H11IN2O6H+, calcd 382.9735); H NMR
(DMSO-d6) δ 11.69 (s, 1H, ex, NH), 8.13 (s, 1H, H6), 5.65 (d, 1H,
ex, J = 4.5 Hz, 3′−OH), 5.40 (s, 1H, H1′), 5.27 (t, 1H, ex, J = 5.4 Hz,
5′−OH), 4.14 (s, 1H, H2′), 3.91 (d, 1H, J = 4.5 Hz, H3′), 3.79−3.82
(d, 1H, J = 8.5 Hz, H5″), 3.68−3.75 (m, 2H, H5′), 3.58−3.62 (d, 1H,
J = 8.5 Hz, H5″); 13C NMR (DMSO-d6) δ 160.5, 149.6, 143.5 (C6),
88.8, 86.4 (C1′), 78.5 (C2′), 70.8 (C5″), 68.4, 68.2 (C3′), 55.3 (C5′).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-
3-(5-iodoracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (3). Diol 2
(5.00 g, 13.0 mmol) was coevaporated with anhydrous pyridine (100
mL) and redissolved in anhydrous pyridine (100 mL). To this was
added 4,4′-dimethoxytrityl chloride (DMTr-Cl, 5.75 g, 16.9 mmol),
and the reaction mixture was stirred at room temperature for 16 h,
whereupon solvent was evaporated off. The residue was dissolved in
CH2Cl2 (300 mL) and washed with saturated aqueous NaHCO3 (300
mL). The aqueous layer was back-extracted with CH2Cl2 (2 × 100
mL), and the combined organic layer was washed with saturated
aqueous NaHCO3 (100 mL), dried (Na2SO4), evaporated to near
dryness, and coevaporated with toluene/absolute EtOH (100 mL, 1/2,
v/v). The resulting residue was purified by column chromatography
(0−5% MeOH in CH2Cl2, v/v) to afford key intermediate 3 (7.52 g,
82%) as a slightly yellow solid material: Rf = 0.5 (5% MeOH in
1
K]+, C33H30N2O8·K+, calcd 621.1634); H NMR (DMSO-d6) δ 11.70
(s, 1H, ex, NH), 7.88 (s, 1H, H6), 7.21−7.45 (m, 9H, Ar), 6.88−6.92
(m, 4H, Ar), 5.70 (d, 1H, ex, J = 4.5 Hz, 3′−OH), 5.45 (s, 1H, H1′),
4.27 (s, 1H, H2′), 4.05 (d, 1H, J = 4.5 Hz, H3′), 3.95 (s, 1H, CH),
3.77 (s, 2H, 2 × H5″), 3.75 (s, 6H, 2 × CH3O), 3.43−3.45 (d, 1H, J =
11.0 Hz, H5′), 3.28−3.31 (d, 1H, J = 11.0 Hz, H5′, overlap with H2O
signal); 13C NMR (DMSO-d6) δ 161.7, 158.1, 149.0, 144.6, 142.2
(C6), 135.3, 135.2, 129.7 (Ar), 129.6 (Ar), 127.9 (Ar), 127.5 (Ar),
126.7 (Ar), 113.3 (Ar), 97.2, 87.5, 86.9 (C1′), 85.7, 83.6, 78.9 (C2′),
76.2, 71.4 (C5″), 69.4 (C3′), 59.0 (C5′), 55.0 (CH3O).
(1R,3R,4R,7S)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-1-(4,4′-
dimethoxytrityloxymethyl)-7-hydroxy-2,5-dioxabicyclo[2.2.1]-
heptane (4c). Nucleoside 3 (0.50 g, 0.73 mmol), Pd(PPh3)4 (90 mg,
0.07 mmol), CuI (30 mg, 0.14 mmol), prop-2-ynyl benzoate46 (180
mg, 1.12 mmol), and Et3N (0.40 mL, 2.84 mmol) in DMF (10 mL)
were reacted as described in the representative Sonogashira protocol,
and the reaction mixture was stirred at room temperature for 12 h.
After workup and purification, nucleoside 4c (0.37 g, 70%) was
obtained as a light brown solid material: Rf = 0.5 (5% MeOH in
CH2Cl2, v/v); ESI-HRMS m/z 739.2289 ([M + Na]+, C41H36N2O10·
+
CH2Cl2, v/v); FAB-HRMS m/z 684.0977 ([M]+, C31H29IN2O8 , calcd
684.0969); H NMR (DMSO-d6) δ 11.74 (s, 1H, ex, NH), 7.96 (s,
1
1H, H6), 7.23−7.45 (m, 9H, Ar), 6.91 (d, 4H, J = 8.5 Hz, Ar), 5.70 (d,
1H, ex, J = 4.5 Hz, 3′−OH), 5.44 (s, 1H, H1′), 4.24 (s, 1H, H2′), 4.07
(d, 1H, J = 4.5 Hz, H3′), 3.74−3.76 (m, 8H, 2 × OCH3, 2 × H5″),
3.39−3.42 (d, 1H, J = 11.0 Hz, H5′), 3.28−3.31 (d, 1H, J = 11.0 Hz,
H5′, overlap with H2O); 13C NMR (DMSO-d6) δ 160.5, 158.1, 158.0,
149.7, 144.6, 142.7 (C6), 135.3, 135.2, 129.7 (Ar), 129.6 (Ar), 127.9
(Ar), 127.5 (Ar), 126.6 (Ar), 113.3 (Ar), 87.5, 86.9 (C1′), 85.6, 78.8
(C2′), 71.3 (C5″), 69.4 (C3′), 68.9, 58.9 (C5′), 55.0 (CH3O).
Representative Protocol for Sonogashira Couplings (4a−j).
Key intermediate 3, Pd(PPh3)4, CuI, and alkyne were added to
anhydrous DMF (quantities and volumes specified below), and the
reaction chamber was degassed and placed under an argon
atmosphere. To this was added Et3N, and the reaction mixture was
stirred in the dark until analytical TLC indicated full conversion of the
starting material (reaction time and temperature specified below),
whereupon solvents were evaporated off. The resulting residue was
taken in up in EtOAc (100 mL) and washed with brine (2 × 50 mL)
and saturated aqueous NaHCO3 (50 mL). The combined aqueous
phase was back-extracted with EtOAc (100 mL), the combined organic
phase dried (Na2SO4) and evaporated to dryness, and the resulting
residue purified by column chromatography (0−5% MeOH in CH2Cl2
(v/v) to afford the desired product.
1
Na, calcd 739.2262); H NMR (DMSO-d6) δ 11.72 (s, 1H, ex, NH),
7.91−7.93 (d, 2H, J = 7.7 Hz, Bzortho), 7.89 (s, 1H, H6), 7.65−7.70 (t,
1H, J = 7.7 Hz, Bzpara), 7.49−7.54 (t, 2H, J = 7.7 Hz, Bzmeta), 7.42−
7.46 (d, 2H, J = 8.5 Hz, DMTr), 7.28−7.34 (m, 6H, DMTr), 7.18−
7.22 (t, 1H, J = 7.5 Hz, DMTr), 6.87−6.91 (2d, 4H, J = 9.0 Hz,
DMTr), 5.74 (d, 1H, ex, J = 4.5 Hz, 3′−OH), 5.42 (s, 1H, H1′), 4.93−
4.97 (d, 1H, J = 16.0 Hz, CH2OBz), 4.86−4.90 (d, 1H, J = 16.0 Hz,
CH2OBz), 4.25 (s, 1H, H2′), 4.10 (d, 1H, J = 4.5 Hz, H3′), 3.78−3.82
(d, 1H, J = 8.0 Hz, H5″), 3.75−3.76 (d, 1H, J = 8.0 Hz, H5″), 3.71 (s,
6H, 2 × CH3O), 3.53−3.56 (d, 1H, J = 11.0 Hz, H5′), 3.27−3.30 (d,
1H, J = 11.0 Hz, H5′, overlap with H2O); 13C NMR (DMSO-d6) δ
164.9, 161.6, 158.1, 158.0, 149.0, 144.6, 142.7 (C6), 135.4, 135.0,
133.5 (CBzpara), 129.7 (DMTr), 129.6 (DMTr), 129.2 (CBzortho),
129.0, 128.7 (CBzmeta), 127.8 (DMTr), 127.5 (DMTr), 126.6
(DMTr), 113.20 (DMTr), 113.17 (DMTr), 96.8, 87.6, 86.9 (C1′),
86.3, 85.6, 79.2, 78.7 (C2′), 71.3 (C5″), 69.4 (C3′), 58.7 (C5′), 54.9
(CH3O), 53.1 (CH2OBz).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(3-trifluoroacetylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo-
[2.2.1]heptane (4d). Nucleoside 3 (0.50 g, 0.73 mmol), Pd(PPh3)4
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(trimethylsilylethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
5055
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Article
(90 mg, 0.07 mmol), CuI (30 mg, 0.14 mmol), 2,2,2-trifluoro-N-
(prop-2-ynyl)acetamide47 (180 mg, 1.46 mmol), and Et3N (0.40 mL,
2.84 mmol) in DMF (10 mL) were reacted as described in the
representative Sonogashira protocol, and the mixture was stirred at
room temperature for 12 h. After workup and purification, nucleoside
4d (0.41 g, 80%) was obtained as a brown solid material: Rf = 0.5 (5%
MeOH in CH2Cl2, v/v); ESI-HRMS m/z 714.2247 ([M+Li]+,
(CH2CONH), 31.2 (CH2), 28.94 (CH2), 28.92 (CH2), 28.8 (CH2),
28.7 (CH2), 28.63 (CH2), 28.60 (CH2), 28.52 (CH2NHCO), 25.0
(CH2CH2CONH), 22.0 (CH2CH3), 13.9 (CH3).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(3-octadecanoylaminopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo-
[2.2.1]heptane (4g). Nucleoside 3 (0.34 g, 0.50 mmol), Pd(PPh3)4
(60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol), N-(prop-2-
ynyl)stearamide (0.28 g, 1.00 mmol), and Et3N (0.30 mL, 2.13
mmol) in DMF (10 mL) were reacted as described in the
representative Sonogashira protocol, and the mixture was stirred at
40 °C for 6 h. After workup and purification, nucleoside 4g (0.29 g,
68%) was obtained as a brown solid material, which was used in the
next step without further purification: Rf = 0.5 (5% MeOH in CH2Cl2,
1
C36H32F3N3O9·Li+, calcd 714.2245); H NMR (DMSO-d6) δ 11.69
(s, 1H, ex, NH(U)), 9.95 (t, 1H, ex, J = 5.5 Hz, NHCH2), 7.78 (s, 1H,
H6), 7.22−7.46 (m, 9H, Ar), 6.90 (dd, 4H, J = 9.0 Hz, 3.5 Hz, Ar),
5.73 (d, 1H, ex, J = 4.5 Hz, 3′−OH), 5.41 (s, 1H, H1′), 4.25 (s, 1H,
H2′), 3.97−4.10 (m, 3H, H3′, CH2NH), 3.79−3.83 (2d, 2H, J = 8.0
Hz, 2 × H5″), 3.74 (s, 6H, 2 × CH3O), 3.56−3.58 (d, 1H, J = 11.0 Hz,
H5′), 3.26−3.28 (d, 1H, J = 11.0 Hz, H5′); 13C NMR (DMSO-d6) δ
161.6, 158.11, 158.06, 155.9 (q, J = 36.1 Hz, COCF3), 149.0, 144.6,
142.1 (C6), 135.4, 134.9, 129.8 (Ar), 129.6 (Ar), 127.8 (Ar), 127.5
(Ar), 126.6 (Ar), 115.6 (q, J = 287 Hz, CF3), 113.22 (Ar), 113.20
(Ar), 97.2, 87.6, 87.2, 86.9 (C1′), 85.6, 78.7 (C2′), 75.4, 71.3 (C5″),
69.6 (C3′), 59.1 (C5′), 55.0 (CH3O), 29.4 (CH2NH); 19F (DMSO-d6,
470 MHz) δ −74.7.
+
v/v); FAB-HRMS m/z 877.4844 ([M]+, C52H67N3O9 , calcd
1
877.4877); H NMR (CDCl3) δ 9.45 (br s, 1H, ex, NH(U)), 8.05
(s, 1H, H6), 7.22−7.50 (m, 9H, Ar), 6.85−6.89 (dd, 4H, J = 9.0 Hz,
1.5 Hz, Ar), 5.56−5.59 (m, 2H, 1 ex, H1′, NHCH2), 4.53 (s, 1H, H2′),
4.29 (s, 1H, H3′), 3.78−4.01 (m, 10H, 2 × H5″, CH2NH, 2 × CH3O),
3.53−3.57 (d, 1H, J = 11.0 Hz, H5′), 3.49−3.52 (d, 1H, J = 11.0 Hz,
H5′), 3.35 (br s, 1H, ex, 3′−OH), 1.85−1.89 (m, 2H, CH2CONH),
1.44−1.51 (m, 2H, CH2CH2CONH), 1.23−1.28 (m, 28H, 14 × CH2),
0.89 (t, 3H, J = 6.5 Hz, CH3); 13C NMR (CDCl3) δ 172.7, 162.1,
158.69, 158.67, 148.6, 144.6, 141.9 (C6), 135.5, 135.4, 130.02 (Ar),
130.01 (Ar), 128.1 (Ar), 128.0 (Ar), 127.0 (Ar), 113.45 (Ar), 113.43
(Ar), 99.1, 89.9, 88.4, 87.4 (C1′), 86.6, 79.1 (C2′), 74.2, 71.8 (C5″),
70.5 (C3′), 58.5 (C5′), 55.3 (CH3O), 36.1 (CH2CONH), 31.9
(CH2), 29.9 (CH2NH), 29.69 (CH2), 29.68 (CH2), 29.66 (CH2), 29.6
(CH2), 29.5 (CH2), 29.4 (CH2), 29.34 (CH2), 29.33 (CH2), 25.4
(CH2CH2CONH), 22.7 (CH2), 14.1 (CH3). A small impurity of
silicon grease was observed at ∼1 ppm in the 13C NMR.48
(1R,3R,4R,7S)-3-[5-(3-(1-Adamantylmethylcarbonyl)amino-
propyn-1-yl)uracil-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-hy-
droxy-2,5-dioxabicyclo[2.2.1]heptane (4e). Nucleoside 3 (0.50 g,
0.73 mmol), Pd(PPh3)4 (90 mg, 0.05 mmol), CuI (30 mg, 0.10
mmol), N-(prop-2-ynyl)-1-adamantaneacetamide (220 mg, 1.00
mmol), and Et3N (0.40 mL, 2.84 mmol) in DMF (10 mL) were
reacted as described in the representative Sonogashira protocol, and
the mixture was stirred at room temperature for 12 h. After workup
and purification, nucleoside 4e (0.43 g, 76%) was obtained as a white
solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS
1
m/z 810.3330 ([M + Na]+, C46H49N3O9·Na+, calcd 810.3361); H
(1R,3R,4R,7S)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)-
uracil-1-yl]-1-(4,4′-dimethoxytrityloxymethyl)-7-hydroxy-2,5-
dioxabicyclo[2.2.1]heptane (4h). Nucleoside 3 (0.34 g, 0.50 mmol),
Pd(PPh3)4 (60 mg, 0.05 mmol), CuI (20 mg, 0.10 mmol),
cholesterylprop-2-ynylcarbamate49 (0.47 g, 1.00 mmol), and Et3N
(0.30 mL, 2.13 mmol) in DMF (8 mL) were reacted as described in
the representative Sonogashira protocol, and the mixture was stirred at
room temperature for 12 h. After workup and purification, nucleoside
4h (0.27 g, 53%) was obtained as a brown solid material, which was
used in the next step without further purification: Rf = 0.5 (5% MeOH
in CH2Cl2, v/v); FAB-HRMS m/z 1046.5560 ([M + Na]+,
NMR (DMSO-d6) δ 11.66 (s, 1H, ex, NH(U)), 8.00 (t, 1H, ex, J = 5.5
Hz, NHCO), 7.75 (s, 1H, H6), 7.42−7.45 (m, 2H, Ar), 7.23−7.34 (m,
7H, Ar), 6.89−6.92 (2d, 4H, J = 9.0 Hz, Ar), 5.72 (d, 1H, ex, J = 4.5
Hz, 3′−OH), 5.42 (s, 1H, H1′), 4.24 (s, 1H, H2′), 4.01 (d, 1H, J = 4.5
Hz, H3′), 3.87−3.93 (dd, 1H, J = 17.5 Hz, 5.5 Hz, CH2NHCO),
3.82−3.87 (dd, 1H, J = 17.5 Hz, 5.5 Hz, CH2NHCO), 3.78−3.82 (2d,
2H, J = 8.2 Hz, H5″), 3.75 (s, 3H, CH3O), 3.74 (s, 3H, CH3O), 3.54−
3.56 (d, 1H, J = 11.0 Hz, H5′), 3.27−3.30 (d, 1H, J = 11.0 Hz, H5′,
overlap with H2O), 1.82−1.87 (m, 5H, 3× ada-CH/CH2CONH),
1.53−1.63 (m, 12H, 6 × ada-CH2); 13C NMR (DMSO-d6) δ 169.6,
161.7, 158.12, 158.07, 149.0, 144.7, 141.5 (C6), 135.4, 134.9, 129.8
(Ar), 129.6 (Ar), 127.9 (Ar), 127.5 (Ar), 126.7 (Ar), 113.24 (Ar),
113.23 (Ar), 97.7, 89.6, 87.5, 86.9 (C1′), 85.6, 78.8 (C2′), 74.2, 71.4
(C5″), 69.6 (C3′), 59.1 (C5′), 54.9 (CH3O), 49.5 (CH2CONH), 42.0
(ada-CH2), 36.4 (ada-CH2), 32.3, 28.4 (CH2NHCO), 28.0 (ada-CH).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-3-[5-(3-dodeca-
noylaminopropyn-1-yl)uracil-1-yl]-7-hydroxy-2,5-dioxabicyclo-
[2.2.1]heptane (4f). Nucleoside 3 (200 mg, 0.29 mmol), Pd(PPh3)4
(34 mg, 0.03 mmol), CuI (11 mg, 0.06 mmol), N-(prop-2-
ynyl)lauroylamide (110 mg, 0.44 mmol), and Et3N (0.18 mL, 1.29
mmol) in DMF (3 mL) were reacted as described in the representative
Sonogashira protocol, and the mixture was stirred at room temperature
for 15 h. After workup and purification, nucleoside 4f (202 mg, 87%)
was obtained as a white solid material: Rf = 0.2 (5% MeOH in CH2Cl2,
v/v); MALDI-HRMS m/z 816.3835 ([M + Na]+, C46H55N3O9·Na+,
1
C62H77N3O10·Na+, calcd 1046.5507); H NMR (CDCl3) δ 9.40 (br
s, 1H, ex, NH(U)), 7.97 (s, 1H, H6), 7.22−7.49 (m, 9H, Ar), 6.87 (d,
4H, J = 9.0 Hz, Ar), 5.58 (s, 1H, H1′), 5.34 (d, 1H, J = 5.0 Hz, HC
C-chol), 4.96 (bs, 1H, ex, NHCH2), 4.53 (s, 1H, H2′), 4.40−4.47 (m,
1H, HC-O-chol) 4.23 (bs, 1H, H3′), 3.83−3.98 (m, 4H, 2 × H5″,
CH2NH), 3.80 (s, 3H, CH3O), 3.79 (s, 6H, 2 × CH3O), 3.58−3.61 (d,
1H, J = 11.0 Hz, H5′), 3.51−3.53 (d, 1H, J = 11.0 Hz, H5′), 3.27 (bs,
1H, ex, 3′-OH), 0.87−2.29 (m, 40 H, chol), 0.69 (s, 3H, CH3-chol);
13C NMR (CDCl3) δ 162.1, 158.63, 158.60, 155.5, 148.7, 144.5, 141.8
(C6), 139.8, 135.44, 135.39, 130.0 (Ar), 128.1 (Ar), 128.0 (Ar), 127.0
(Ar), 122.5 (CH, chol), 113.4 (Ar), 99.2, 90.1, 88.4, 87.4 (C1′),
86.6, 79.1 (C2′), 74.7 (OCH-chol), 74.1, 71.9 (C5″), 70.6 (C3′), 58.6
(C5′), 56.7 (CH-chol), 56.2 (CH-chol), 55.2 (CH3O), 50.0 (CH-
chol), 42.3, 39.8 (CH2-chol), 39.5 (CH2-chol), 38.5 (CH2-chol), 37.0
(CH2-chol), 36.5, 36.2 (CH2-chol), 35.8 (CH-chol), 31.9 (CH-chol/
CH2NH), 28.2 (CH2-chol), 28.1 (CH2-chol), 28.0 (CH-chol), 24.3
(CH2-chol), 23.8 (CH2-chol), 22.8 (CH3-chol), 22.5 (CH3-chol), 21.0
(CH2-chol), 19.3 (CH3-chol), 18.7 (CH3-chol), 11.8 (CH3-chol).
Signals at 41.4, 29.0, 22.6, 20.4, 19.4, 14.3, and 11.4 ppm, presumably
arising from a small contamination of unreacted cholesterylprop-2-
ynylcarbamate, were also observed in the 13C NMR spectrum.
1
calcd 816.3831); H NMR (DMSO-d6) δ 11.67 (s, 1H, ex, NH(U)),
8.08 (t, 1H, J = 5.4 Hz, NHCH2), 7.76 (s, 1H, H6), 7.22−7.46 (m, 9H,
Ar), 6.87−6.96 (m, 4H, Ar), 5.72 (d, 1H, ex, J = 4.7 Hz, 3′−OH), 5.42
(s, 1H, H1′), 4.25 (s, 1H, H2′), 4.03 (d, 1H, J = 4.7 Hz, H3′), 3.88−
3.94 (dd, 1H, J = 12.4 Hz, 5.4 Hz, CH2NHCO), 3.81−3.88 (dd, 1H, J
= 12.4 Hz, 5.4 Hz, CH2NHCO), 3.78−3.82 (2d, 2H, J = 8.0 Hz, H5″),
3.75 (s, 6H, 2 × CH3O), 3.55−3.57 (d, 1H, J = 10.9 Hz, H5′), 3.27−
3.30 (d, 1H, J = 10.9 Hz, H5′, overlap with H2O), 2.05 (t, 2H, J = 7.4
Hz,CH2CONH), 1.43−1.48 (m, 2H, CH2CH2CONH), 1.19−1.28
(m, 16H, 8 × CH2), 0.85 (t, 3H, J = 7.0 Hz, CH3); 13C NMR
(DMSO) δ 171.7, 161.7, 158.11, 158.06, 149.0, 144.6, 141.6 (C6),
135.4, 134.9, 129.8 (Ar), 129.6 (Ar), 127.9 (Ar), 127.5 (Ar), 126.6
(Ar), 113.22 (Ar), 113.21 (Ar), 97.7, 89.5, 87.5, 86.9 (C1′), 85.6, 78.8
(C2′), 74.3, 71.4 (C5″), 69.6 (C3′), 59.0 (C5′), 55.0 (CH3O), 35.0
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(2-(1-pyrenyl)ethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(4i). Nucleoside 3 (0.34 g, 0.50 mmol), Pd(PPh3)4 (60 mg, 0.05
mmol), CuI (20 mg, 0.10 mmol), 1-ethynylpyrene50 (0.28 g, 1.00
mmol), and Et3N (0.30 mL, 2.84 mmol) in DMF (10 mL) were
reacted as described in the representative Sonogashira protocol, and
the mixture was stirred at room temperature for 12 h. Following
workup and purification, nucleoside 4i (0.31 g, 80%) was obtained as a
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The Journal of Organic Chemistry
Article
slightly yellow solid, which was used in the next step without further
purification: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS m/
z 805.2554 ([M + Na]+, C49H38N2O8·Na+, calcd 805.2520); 1H NMR
(DMSO-d6) δ 11.89 (s, 1H, ex, NH), 8.35 (d, 1H, J = 8.5 Hz, Ar),
8.31−8.34 (d, 1H, J = 8.5 Hz, Ar), 8.25−8.28 (d, 1H, J = 8.0 Hz, Ar),
8.14−8.23 (m, 4H, H6, Ar), 8.09−8.12 (ap t, 1H, J = 8.0 Hz, Ar), 7.93
(d, 1H, J = 8.5 Hz, Ar), 7.67 (d, 1H, J = 8.0 Hz, Ar), 7.49−7.50 (m,
2H, Ar), 7.33−7.38 (m, 4H, Ar), 7.26−7.30 (ap t, 2H, J = 7.5 Hz, Ar),
7.03−7.06 (ap t, 1H, J = 7.5 Hz, Ar), 6.78−6.85 (m, 4H, Ar), 5.78 (d,
1H, ex, J = 4.5 Hz, 3′-OH), 5.53 (s, 1H, H1′), 4.34 (s, 1H, H2′), 4.24
(d, 1H, J = 4.5 Hz, H3′), 3.78−3.82 (2d, 2H, J = 7.5 Hz, H5″), 3.43−
3.52 (m, 7H, OCH3, H5′), 3.38−3.42 (d, 1H, J = 11.0 Hz, H5′); 13C
NMR (DMSO-d6) δ 161.7, 158.02, 157.95, 149.1, 144.4, 141.4 (C6),
135.4, 130.8, 130.7, 130.6, 130.4, 129.6 (Ar), 129.5 (Ar), 128.8 (Ar),
128.2 (Ar), 128.1 (Ar), 127.8 (Ar), 127.7 (Ar), 127.1 (Ar), 126.63
(Ar), 126.58 (Ar), 125.7 (Ar), 125.6 (Ar), 124.8 (Ar), 124.5 (Ar),
123.5, 123.3, 116.9, 113.17 (Ar), 113.16 (Ar), 98.2, 91.3, 88.2, 87.8,
87.2 (C1′), 85.6, 78.8 (C2′), 71.4 (C5″), 69.4 (C3′), 58.7 (C5′), 54.7
(OCH3), 54.6 (OCH3). A trace of pyridine was observed in the 13C
NMR.48
(s, 1H, H1′), 4.43 (s, 1H, H2′), 4.13 (d, 1H, J = 4.5 Hz, H3′), 3.94−
3.97 (d, 1H, J = 9.0 Hz, H5″), 3.86−3.90 (d, 1H, J = 9.0 Hz, H5″),
3.681 (s, 3H, CH3O), 3.675 (s, 3H, CH3O), 3.57−3.60 (d, 1H, J =
11.0 Hz, H5′), 3.34−3.37 (m, 1H, J = 11.0 Hz, H5′); 13C NMR
(DMSO-d6) δ 161.3, 158.05, 158.02, 149.4, 144.6, 139.4, 135.3, 135.2,
135.0 (C6), 131.6, 130.6, 130.14, 130.09, 129.7 (Ar), 129.6 (Ar), 129.5
(Ar), 128.7 (Ar), 127.8 (Ar), 127.6 (Ar), 127.0 (Ar), 126.6 (Ar), 126.5
(Ar), 126.1 (Ar), 125.3, 125.1 (Ar), 124.8 (CH-Tz), 124.0, 123.7 (Ar),
123.3, 120.9 (Ar), 113.24 (Ar), 113.19 (Ar), 104.3, 87.6, 87.2 (C1′),
85.6, 79.0 (C2′), 71.5 (C5″), 70.0 (C3′), 59.5 (C5′), 54.9 (CH3O).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-
3-[5-(1-(pyren-1-ylmethyl)-1H-1,2,3-triazol-4-yl)-uracil-1-yl]-
2,5-dioxabicyclo[2.2.1]heptane (4l). To a solution of nucleoside
4b (0.33 g, 0.56 mmol) and 1-azidomethylpyrene27 (200 mg, 0.78
mmol) in THF/H2O/t-BuOH (10 mL, 3/1/1, v/v/v) were added
aqueous sodium ascorbate (1 M, 1.00 mL, 1.00 mmol) and aqueous
CuSO4 (7.5%, w/v, 1.00 mL, 0.30 mmol). The reaction mixture was
stirred at room temperature for 2 h, whereupon it was taken up in
EtOAc (50 mL) and brine (50 mL). The layers were separated, and
the organic phase was washed with saturated aqueous NaHCO3 (50
mL). The combined aqueous phase was back-extracted with EtOAc
(50 mL). The combined organic phase was dried (Na2SO4) and
evaporated to dryness and the resulting residue purified by column
chromatography (0−75% EtOAc in petroleum ether, v/v) to afford
nucleoside 4l (0.43 g, 91%) as a slightly yellow solid material: Rf = 0.4
(70% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 862.2869 ([M
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-3-[5-
(2-(3-perylenyl)ethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(4j). Nucleoside 3 (0.50 g, 0.73 mmol), Pd(PPh3)4 (90 mg, 0.07
mmol), CuI (30 mg, 0.14 mmol), 3-ethynylperylene51 (0.27 g, 1.00
mmol), and Et3N (0.40 mmol, 2.84 mmol) in DMF (10 mL) were
reacted as described in the representative Sonogashira protocol, and
the mixture was stirred at room temperature for 12 h. After workup
and purification, nucleoside 4j (0.49 g, 80%) was obtained as a brown
solid material: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS
1
+ Na]+, C50H41N5O8·Na+, calcd 862.2847); H NMR (DMSO-d6) δ
11.70 (s, 1H, ex, NH), 8.56 (d, 1H, J = 9.0 Hz, Ar), 8.44 (s, 1H, H-
Tz), 8.29−8.36 (m, 5H, H6, Ar), 8.20−8.23 (d, 1H, J = 9.0 Hz, Ar),
8.17−8.20 (d, 1H, J = 9.0 Hz, Ar), 8.09−8.12 (t, 1H, J = 8.0 Hz, Ar),
8.07 (d, 1H, J = 8.0 Hz, Ar), 7.39−7.42 (d, 2H, J = 7.5 Hz, Ar), 7.22−
7.33 (m, 6H, Ar), 7.09−7.12 (t, 1H, J = 7.5 Hz, Ar), 6.88 (d, 4H, J =
8.0 Hz, Ar), 6.40 (s, 2H, CH2Py), 5.69 (d, 1H, ex, J = 4.5 Hz, 3′-OH),
5.55 (s, 1H, H1′), 4.32 (s, 1H, H2′), 3.91−3.96 (m, 2H, H3′, H5″),
3.81−3.84 (d, 1H, J = 8.0 Hz, H5″), 3.70 (s, 3H, CH3O), 3.68 (s, 3H,
CH3O), 3.50−3.54 (d, 1H, J = 11.0 Hz, H5′), 3.25−3.29 (d, 1H, J =
11.0 Hz, H5′); 13C NMR (DMSO-d6) δ 161.2, 158.1, 158.0, 149.2,
144.7, 138.9, 135.1, 134.2 (C6), 131.0, 130.7, 130.1, 129.7 (Ar), 129.6
(Ar), 129.1, 128.4, 128.2 (Ar), 127.8 (Ar), 127.6 (Ar), 127.5 (Ar),
127.2 (Ar), 126.52 (Ar), 126.45 (Ar), 125.7 (Ar), 125.5 (Ar), 125.0
(Ar), 124.0, 123.7, 122.7 (Ar), 122.4 (CH-Tz), 113.3 (Ar), 113.2 (Ar),
104.5, 87.5, 87.1 (C1′), 85.6, 79.0 (C2′), 71.6 (C5″), 70.0 (C3′), 59.8
(C5′), 54.9 (CH3O), 54.8 (CH3O), 50.7 (CH2Py).
Representative Procedure for O3′-Phosphitylation. Alcohols
5b−l were dried by coevaporation with anhydrous 1,2-dichloroethane
and dissolved in anhydrous CH2Cl2. To this were added anhydrous
N,N′-diisopropylethylamine (DIPEA) and 2-cyanoethyl-N,N-diisopro-
pylchlorophosphoramidite (PCl-reagent) (quantities and volumes
specified below), and the reaction mixture was stirred at room
temperature until analytical TLC indicated complete conversion (2 h
unless otherwise mentioned). Unless otherwise mentioned, the
reaction mixture was diluted with CH2Cl2 (25 mL) and washed with
aqueous NaHCO3 (2 × 10 mL), the combined aqueous phases were
back-extracted with CH2Cl2 (2 × 10 mL), and the combined organic
phases were dried (Na2SO4) and evaporated to dryness. Regardless of
the workup procedure, the resulting residue was purified by silica gel
column chromatography (typically 0−4% MeOH/CH2Cl2, v/v) and
subsequent trituration from CH2Cl2 and petroleum ether to provide
the target phosphoramidites.
1
m/z 855.2675 ([M + Na]+, C53H40N2O8·Na+, calcd 855.2677); H
NMR (DMSO-d6) δ 11.86 (s, 1H, ex, NH), 8.34−8.40 (m, 3H, Ar),
8.22 (d, 1H, J = 8.0 Hz, Ar), 8.13 (s, 1H, H6), 8.02 (d, 1H, J = 8.5 Hz,
Ar), 7.79−7.85 (m, 2H, Ar), 7.55 (dt, 2H, J = 8.0 Hz, 2.0 Hz, Ar),
7.48−7.51 (m, 2H, Ar), 7.28−7.37 (m, 7H, Ar), 7.22 (d, 1H, J = 8.0
Hz, Ar), 7.09−7.12 (t, 1H, J = 7.5 Hz, Ar), 6.83−6.88 (m, 4H, Ar),
5.77 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.51 (s, 1H, H1′), 4.33 (s, 1H,
H2′), 4.22 (d, 1H, J = 4.5 Hz, H3′), 3.77−3.83 (m, 2H, 2 × H5″), 3.59
(s, 3H, OCH3), 3.56 (s, 3H, OCH3), 3.48−3.51 (d, 1H, J = 11.0 Hz,
H5′), 3.35−3.39 (d, 1H, J = 11.0 Hz, H5′); 13C NMR (DMSO-d6) δ
161.7, 158.05, 158.00, 149.0, 144.4, 141.3 (C6), 135.43, 135.37, 134.2,
133.6, 130.9, 130.7, 130.4 (Ar), 130.1, 129.9, 129.6 (Ar), 129.5 (Ar),
128.5 (Ar), 128.2 (Ar), 127.9 (Ar), 127.7 (Ar), 127.60, 127.57, 127.47
(Ar), 127.0 (Ar), 126.9 (Ar), 126.7 (Ar), 125.7 (Ar), 121.5 (Ar),
121.2, 121.1 (Ar), 120.0 (Ar), 119.4, 113.2 (Ar), 98.1, 90.9, 88.5, 87.7,
87.1 (C1′), 85.6, 78.8 (C2′), 71.4 (C5″), 69.4 (C3′), 58.7 (C5′), 54.80
(CH3O), 54.75 (CH3O).
(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-
3-[5-(1-(1-pyrenyl)-1H-1,2,3-triazol-4-yl)-uracil-1-yl]-2,5-
dioxabicyclo[2.2.1]heptane (4k). To a solution of nucleoside 4b
(0.25 g, 0.36 mmol) and 1-pyrenyl azide26 (110 mg, 0.45 mmol) in
THF/H2O/t-BuOH (10 mL, 3/1/1, v/v/v) were added aqueous
sodium ascorbate (1 M, 0.70 mL, 0.70 mmol) and aqueous CuSO4
(7.5%, w/v, 0.65 mL, 0.19 mmol). The solution was stirred at room
temperature for 2 h, whereupon it was taken up in EtOAc (50 mL)
and brine (50 mL). The layers were separated, and the organic phase
was washed with saturated aqueous NaHCO3 (50 mL). The combined
aqueous phase was back-extracted with EtOAc (50 mL). The
combined organic phase was then dried (Na2SO4) and evaporated
to dryness and the resulting residue purified by column chromatog-
raphy (0−75% EtOAc in petroleum ether, v/v) to afford nucleoside 4k
(230 mg, 76%) as a slightly yellow solid material: Rf = 0.4 (70% EtOAc
in petroleum ether, v/v); ESI-HRMS m/z 848.2712 ([M + Na]+,
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-(5-ethynyluracil-1-yl)-2,5-
dioxabicyclo[2.2.1]heptane (5b). Nucleoside 4b (0.34 g 0.58 mmol),
DIPEA (0.50 mL, 2.90 mmol), PCl-reagent (0.20 mL, 0.87 mmol),
and anhydrous CH2Cl2 (10 mL) were mixed, reacted, worked up, and
purified as described in the representative protocol to provide
nucleoside 5b (0.38 g, 83%) as a white foam: Rf = 0.5 (2% MeOH
in CH2Cl2, v/v); ESI-HRMS m/z 805.2973 ([M + Na]+,
C42H47N4O9P·Na+, calcd 805.2958); 31P NMR (CDCl3) δ 149.8,
149.3.
1
C49H39N5O8·Na+, calcd 848.2691); H NMR (DMSO-d6) δ 11.85 (s,
1H, ex, NH), 8.84 (s, 1H, H-Tz), 8.58 (s, 1H, H6), 8.48 (d, 1H, J = 8.0
Hz, Ar), 8.44−8.47 (d, 1H, J = 7.5 Hz, Ar), 8.40−8.43 (d, 1H, J = 8.0
Hz, Ar), 8.35−8.38 (d, 1H, J = 9.0 Hz, Ar), 8.32−8.35 (d, 1H, J = 9.0
Hz, Ar), 8.30 (d, 1H, J = 9.0 Hz, Ar), 8.16−8.20 (overlapping d and t,
2H, Ar), 7.79 (d, 1H, J = 8.0 Hz, Ar), 7.45−7.48 (d, 2H, J = 7.5 Hz,
Ar), 7.28−7.39 (m, 6H, Ar), 7.17−7.20 (t, 1H, J = 7.5 Hz, Ar), 6.88−
6.93 (2d, 4H, J = 9.0 Hz, Ar), 5.79 (d, 1H, ex, J = 4.5 Hz, 3′-OH), 5.64
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Article
(1R,3R,4R,7S)-3-[5-(3-Benzoyloxypropyn-1-yl)uracil-1-yl]-7-[2-
cyanoethoxy(diisopropylamino)phosphinoxy]-1-(4,4′-dimethoxytri-
tyloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (5c). Nucleoside 4c
(0.30 g, 0.42 mmol), DIPEA (300 μL, 1.67 mmol), PCl-reagent (121
μL, 0.54 mmol), and anhydrous CH2Cl2 (5 mL) were mixed and
reacted (5 h) as described above. At this point the reaction mixture
was concentrated to one-third volume and diluted with diethyl ether
(100 mL) and the organic phase sequentially washed with H2O (35
mL), H2O/DMF (70 mL, 1/1, v/v), H2O (35 mL), and brine (35
mL). The organic phase was evaporated to dryness and the resulting
residue purified as described in the representative protocol to provide
nucleoside 5c (150 mg, 43%) as a white foam: Rf = 0.6 (5% MeOH in
CH2Cl2, v/v); ESI-HRMS m/z 939.3356 ([M + Na]+, C50H53N4O11P·
Na+, calcd 939.3341); 31P NMR (CDCl3) δ 149.8, 149.3.
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-trifluoroacetylami-
nopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5d). Nu-
cleoside 4d (0.37 g 0.52 mmol), DIPEA (0.44 mL, 2.52 mmol), PCl-
reagent (0.18 mL, 0.78 mmol), and anhydrous CH2Cl2 (10 mL) were
mixed, reacted, worked up, and purified as described in the
representative protocol to provide nucleoside 5d (0.39 g, 80%) as a
white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/z
930.3068 ([M + Na]+, C45H49F3N5O10P·Na+, calcd 930.3061); 31P
NMR (CDCl3) δ 149.7, 149.1.
(1R,3R,4R,7S)-3-[5-(3-(1-Adamantylmethylcarbonyl)amino-
propyn-1-yl)uracil-1-yl]-7-[2-cyanoethoxy(diisopropylamino)-
phosphinoxy]-1-(4,4′-dimethoxytrityloxymethyl)-2,5-dioxabicyclo-
[2.2.1]heptane (5e). Nucleoside 4e (204 mg, 0.26 mmol), DIPEA
(184 μL, 1.06 mmol), and PCl-reagent (106 μL, 0.48 mmol) in
anhydrous CH2Cl2 (4 mL) were mixed and reacted as described in the
representative protocol. At this point, ice-cold EtOH (1 mL) was
added and the solvents were evaporated off. Purification as described
in the representative protocol provided nucleoside 4e (190 mg, 74%)
as a slightly yellow foam: Rf = 0.4 (5% MeOH in CH2Cl2, v/v);
MALDI-HRMS m/z 1010.4408 ([M + Na]+, C55H66N5O10P·Na+,
calcd 1010.4440); 31P NMR (CDCl3) δ 149.8, 149.2. A minor
impurity at ∼14 ppm was observed.
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-dodecanoylamino-
propyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5f). Nu-
cleoside 4f (175 mg, 0.22 mmol), DIPEA (154 μL, 0.88 mmol), N-
methylimidazole (14 μL, 0.18 mmol), PCl-reagent (75 μL, 0.33
mmol), and anhydrous CH2Cl2 (3 mL) were mixed and reacted (2.5
h). The solvents were evaporated off, and the resulting residue was
purified as described in the representative protocol to provide
nucleoside 5f (183 mg, 83%) as a white foam: Rf = 0.4 (4% MeOH
in CH2Cl2, v/v); MALDI-HRMS m/z 1016.4983 ([M + Na]+,
C55H72N5O10P·Na+, calcd 1016.4909); 31P NMR (CDCl3) δ 149.8,
149.2.
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(3-octadecanoylami-
nopropyn-1-yl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5g). Nu-
cleoside 4g (0.25 g, 0.28 mmol), DIPEA (0.24 mL, 1.37 mmol), PCl-
reagent 0.10 mL, 0.42 mmol), and anhydrous CH2Cl2 (10 mL) were
mixed, reacted, worked up, and purified as described in the
representative protocol to provide nucleoside 5g (180 mg, 60%) as
a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/z
1100.5836 ([M + Na]+, C61H84N5O10P·Na+, calcd 1100.5848); 31P
NMR (CDCl3) δ 149.8, 149.2.
(1R,3R,4R,7S)-3-[5-(3-Cholesterylcarbonylaminopropyn-1-yl)-
uracil-1-yl]-7-[2-cyanoethoxy(diisopropylamino)phosphinoxy]-1-
(4,4′-dimethoxytrityloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane
(5h). Nucleoside 4h (240 mg, 0.23 mmol), DIPEA (0.19 mL, 1.08
mmol), PCl-reagent (0.08 mL, 0.34 mmol), and anhydrous CH2Cl2
(10 mL) were mixed, reacted, worked up, and purified as described in
the representative protocol to provide nucleoside 5h (190 mg, 66%) as
a white foam: Rf = 0.5 (2% MeOH in CH2Cl2, v/v); ESI-HRMS m/z
1246.6571 ([M + Na]+, C71H94N5O11P·Na+, calcd 1246.6579); 31P
NMR (CDCl3) δ 149.8, 149.3.
uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5i). Nucleoside 4i (0.20
g, 0.26 mmol), DIPEA (225 μL, 1.28 mmol), PCl-reagent (114 μL,
0.51 mmol), and anhydrous CH2Cl2 (4 mL) were mixed and reacted
(4.5 h) as described in the representative protocol. Solvents were
evaporated off, and the resulting residue was purified as described in
the representative protocol to provide nucleoside 5i (188 mg, 75%) as
a pale yellow foam: Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-
HRMS m/z 1005.3661 ([M + Na]+, C58H55N4O9P·Na+, calcd
1005.3606); 31P NMR (CDCl3) δ 149.7, 149.3.
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(2-(3-perylenyl)-
ethynyl)uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5j). Nucleoside
4j (0.47 g, 0.56 mmol), DIPEA (0.40 mL, 2.26 mmol), PCl-reagent
(165 μL, 0.73 mmol), and anhydrous CH2Cl2 (4 mL) were mixed,
reacted (3 h), worked up, and purified as described in the
representative protocol to provide nucleoside 5j (0.43 g, 78%) as a
yellow foam: Rf = 0.4 (4% MeOH in CH2Cl2, v/v); ESI-HRMS m/z
1055.3751 ([M + Na]+, C62H57N4O9P·Na+, calcd 1055.3763); 31P
NMR (CDCl3) δ 149.7, 149.3
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(1-(1-pyrenyl)-1H-
1,2,3-triazol-4-yl)-uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane (5k).
Nucleoside 4k (180 mg, 0.22 mmol), DIPEA (160 μL, 0.88 mmol),
PCl-reagent (63 μL, 0.28 mmol), and anhydrous CH2Cl2 (1.5 mL)
were mixed and reacted (2.5 h) as described in the representative
protocol. At this point, the reaction mixture was diluted with EtOAc
(20 mL) and washed with H2O (2 × 25 mL). The organic phase was
dried (Na2SO4) and evaporated to dryness and the resulting residue
purified as described in the representative protocol to provide
nucleoside 5k (184 mg, 82%) as a white foam: Rf = 0.7 (5% MeOH
in CH2Cl2, v/v); ESI-HRMS m/z 1048.3789 ([M + Na]+,
C58H56N7O9P·Na+, calcd 1048.3769); 31P NMR (CDCl3) δ 149.8,
149.1.
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(1-(pyren-1-ylmethyl)-
1H-1,2,3-triazol-4-yl)-uracil-1-yl]-2,5-dioxabicyclo[2.2.1]heptane
(5l). Nucleoside 4l (0.41 g, 0.49 mmol), DIPEA (345 μL, 1.95 mmol),
PCl-reagent (165 μL, 0.73 mmol), and anhydrous CH2Cl2 (5 mL)
were mixed, reacted (2.5 h), worked up, and purified as described in
the representative protocol to provide nucleoside 5l (190 mg, 40%) as
a white foam: Rf = 0.5 (3% MeOH in CH2Cl2, v/v); ESI-HRMS m/z
1062.3909 ([M + Na]+, C59H58N7O9P·Na+, calcd 1062.3934); 31P
NMR (CDCl3) δ 149.6, 149.1.
Synthesis and Purification of ONs. L1−4, K1−4, N1−4, Q1−4,
S1−4, and V5 were prepared and characterized with respect to identity
(MALDI-MS) and purity (>80%, ion-pair reverse-phase HPLC) in
previous studies.22,37 All of the other ONs were synthesized, worked
up, purified, and characterized essentially as previously described.37
Briefly, ONs were synthesized on a 0.2 μmol scale using an automated
DNA synthesizer and long chain alkyl amine controlled pore glass
columns with a pore size of 500 Å. Standard reagents were used. The
following hand-coupling conditions were employed to incorporate
monomers K−Z into ONs, which generally resulted in coupling yields
in excess of 95% (coupling time; activator; phosphoramidite solvent):
monomers K/L/M/N/O/Q/S/W/Z (15 min; 0.25 M 4,5-dicyanoi-
midazole in CH3CN; CH3CN), monomer P (15 min; 0.25 M 4,5-
dicyanoimidazole in CH3CN; CH2Cl2), monomer V (30 min; 0.25 M
4,5-dicyanoimidazole in CH3CN; CH2Cl2), and monomers X/Y/Z
(15 min; 0.25 M 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole52 in
CH3CN; CH2Cl2). ONs were cleaved from the solid support and
protecting groups removed through treatment with concentrated
aqueous ammonia (55 °C, 24 h). ONs were purified by ion-pair
reverse-phase HPLC (XTerra MS C18 column) using a gradient of
0.05 M triethylammonium acetate in water and 25% water in CH3CN,
followed by detritylation (80% aqueous AcOH) and precipitation
(NaOAc/NaClO4/acetone, −18 °C for 12−16 h). The identity of all
synthesized ONs was verified by MALDI MS analysis (Table S1,
Supporting Information) recorded in positive ion mode on a
quadrupole time-of-flight tandem mass spectrometer equipped with
(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphin-
oxy]-1-(4,4′-dimethoxytrityloxymethyl)-3-[5-(2-(1-pyrenyl)ethynyl)-
5058
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Article
a MALDI source. Purity (>80%) was verified by ion-pair reverse-phase
HPLC running in analytical mode.
Preliminary synthetic efforts by T. Santhosh Kumar (University
of Southern Denmark) are appreciated.
Biophysical Characterization Studies. Thermal denaturation
temperatures and steady-state fluorescence emission spectra were
determined essentially as previously described.37 Briefly, thermal
denaturation temperatures were determined as the maximum of the
first derivative of the thermal denaturation curve (A260 vs T) recorded
in medium salt buffer (Tm buffer: 110 mM NaCl, 0.1 mM EDTA, pH
adjusted with 10 mM Na2HPO4/NaH2PO4). A temperature ramp of
0.5 °C/min was used in all experiments. Reported thermal
denaturation temperatures are an average of at least two experiments
within 1.0 °C.
Thermodynamic parameters for duplex formation were determined
through baseline fitting of denaturation curves (van’t Hoff analysis)
using software provided with the UV/vis spectrometer. Bimolecular
reactions, two-state melting behavior, and a heat capacity change of
ΔCp = 0 upon hybridization were assumed. A minimum of two
experimental denaturation curves were each analyzed to minimize
errors arising from baseline choice. Averages are listed.
3′-Exonuclease degradation studies were performed by observing
the change in absorbance at 260 nm and 37 °C as a function of time
for a solution of ONs (3.3 μM) in magnesium buffer (600 μL, 50 mM
Tris-HCl, 10 mM MgCl2, pH 9.0) to which SVPDE (snake venom
phosphodiesterase) dissolved in H2O was added (12 μL, 0.52 μg, 0.03
U).
Steady-state fluorescence emission spectra were recorded using the
same buffers and ON concentrations (1.0 μM) as in thermal
denaturation studies. Fluorescence emission spectra were recorded at
5 °C to ensure maximum hybridization. Deoxygenation was
deliberately not applied to the samples, since the scope of the work
was to determine fluorescence under aerated conditions prevailing in
bioassays. Steady-state fluorescence emission spectra were obtained as
an average of five scans using λex 344 nm for V/Y/Z-modified ONs, λex
375 nm for W-modified ONs, λex 448 nm for X-modified ONs,
excitation slit 5.0 nm, emission slit 5.0 nm, and a scan speed of 600
nm/min.
REFERENCES
■
(1) For recent reviews on conformationally restricted nucleotides, see
e.g.: (a) Herdewijn, P. Chem. Biodiv. 2010, 7, 1−59. (b) Obika, S.;
Abdur Rahman, S. M.; Fujisaka, A.; Kawada, Y.; Baba, T.; Imanishi, T.
Heterocycles 2010, 81, 1347−1392. (c) Prakash, T. P. Chem. Biodiv.
2011, 8, 1616−1641. (d) Zhou, C.; Chattopadhyaya, J. Chem. Rev.
2012, 112, 3808−3832.
(2) For recent representative examples, see: (a) Seth, P. P.; Vasquez,
G.; Allerson, C. A.; Berdeja, A.; Gaus, H.; Kinberger, G. A.; Prakash, T.
P.; Migawa, M. T.; Bhat, B.; Swayze, E. E. J. Org. Chem. 2010, 75,
1569−1581. (b) Li, Q.; Yuan, F.; Zhou, C.; Plashkevych, O.;
Chattopadhyaya, J. J. Org. Chem. 2010, 75, 6122−6140. (c) Liu, Y.;
Xu, J.; Karimiahmadabadi, M.; Zhou, C.; Chattopadhyaya, J. J. Org.
Chem. 2010, 75, 7112−7128. (d) Upadhayaya, R.; Deshpande, S. A.;
Li, Q.; Kardile, R. A.; Sayyed, A. Y.; Kshirsagar, E. K.; Salunke, R. V.;
Dixit, S. S.; Zhou, C.; Foldesi, A.; Chattopadhyaya, J. J. Org. Chem.
2011, 76, 4408−4431. (e) Shrestha, A. R.; Hari, Y.; Yahara, A.; Osawa,
T.; Obika, S. J. Org. Chem. 2011, 76, 9891−9899. (f) Hanessian, S.;
Schroeder, B. R.; Giacometti, R. D.; Merner, B. L.; Østergaard, M. E.;
Swayze, E. E.; Seth, P. P. Angew. Chem., Int. Ed. 2012, 51, 11242−
11245. (g) Madsen, A. S.; Wengel, J. J. Org. Chem. 2012, 77, 3878−
3886. (h) Haziri, A. I.; Leumann, C. J. J. Org. Chem. 2012, 77, 5861−
5869. (i) Gerber, A.-B.; Leumann, C. J. Chem. Eur. J. 2013, 19, 6990−
7006. (j) Morihiro, K.; Kodama, T.; Kentefu; Moai, Y.; Veedu, R. N.;
Obika, S. Angew. Chem., Int. Ed. 2013, 52, 5074−5078. (k) Hari, Y.;
Osawa, T.; Kotobuki, Y.; Yahara, A.; Shrestha, A. R.; Obika, S. Bioorg.
Med. Chem. 2013, 21, 4405−4412. (l) Hari, Y.; Morikawa, T.; Osawa,
T.; Obika, S. Org. Lett. 2013, 15, 3702−3705. (m) Migawa, M. T.;
Prakash, T. P.; Vasquez, G.; Seth, P. P.; Swayze, E. E. Org. Lett. 2013,
15, 4316−4319. (n) Hanessian, S.; Schroeder, B. R.; Merner, B. L.;
Chen, B.; Swayze, E. E.; Seth, P. P. J. Org. Chem. 2013, 78, 9051−9063.
(o) Hanessian, S.; Wagger, J.; Merner, B. L.; Giacometti, R. D.;
Østergaard, M. E.; Swayze, E. E.; Seth, P. P. J. Org. Chem. 2013, 78,
9064−9075.
ASSOCIATED CONTENT
■
(3) Eschenmoser, A. Science 1999, 284, 2118−2124.
S
* Supporting Information
(4) Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Van Aerschot, A.;
Seela, F.; Herdewijn, P. Chem. Eur. J. 1997, 3, 110−120.
(5) Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.;
Hendrix, C.; Rosemeyer, H.; Seela, F.; Van Aerschot, A.; Herdewijn, P.
J. Am. Chem. Soc. 2000, 122, 8595−8602.
Text, tables, and figures giving general experimental details,
NMR spectra for all new compounds, MS data for all new
modified ONs, additional Tm data, 3′-exonuclease degradation
and fluorescence data, and structures of modified DNA
monomers. This material is available free of charge via the
Internet at http://pubs.acs.org.
(6) Bolli, M.; Trafelet, H. U.; Leumann, C. Nucleic Acids Res. 1996,
24, 4660−4667.
(7) Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124,
5993−6002.
AUTHOR INFORMATION
(8) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
Commun. 1998, 455−456.
■
Corresponding Author
*E-mail for P.J.H.: hrdlicka@uidaho.edu.
(9) Kaur, H.; Babu, B. R.; Maiti, S. Chem. Rev. 2007, 107, 4672−
4697.
Notes
(10) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-I.; Morio, K.-I.; Doi,
T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401−5404.
(11) Kool, E. T. Chem. Rev. 1997, 97, 1473−1487.
The authors declare no competing financial interest.
(12) (a) Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo, P.
B. Nucleic Acids Res. 2008, 36, 5123−5138. (b) Bennett, C. F.; Swayze,
E. E. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259−293. (c) Østergaard,
M. E.; Hrdlicka, P. J. Chem. Soc. Rev. 2011, 40, 5771−5788. (d) Watts,
J. K.; Corey, D. R. J. Pathol. 2012, 226, 365−379. (e) Matsui, M.;
Corey, D. R. Drug Discuss. Today 2012, 17, 443−450. (f) Dong, H.;
Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. Chem. Rev. 2013, 113,
6207−6233.
(13) (a) Wahlestedt, C.; Salmi, P.; Good, L.; Kela, J.; Johnsson, T.;
Hokfelt, T.; Broberger, C.; Porreca, F.; Lai, J.; Ren, K. K.; Ossipov, M.;
Koshkin, A.; Jacobsen, N.; Skouv, J.; Oerum, H.; Jacobsen, M. H.;
Wengel, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5633−5638.
(b) Graziewicz, M. A.; Tarrant, T. K.; Buckley, B.; Roberts, J.;
Fulton, L.; Hansen, H.; Ørum, H.; Kole, R.; Sazani, P. Mol. Ther. 2008,
16, 1316−1322. (c) Straarup, E. M.; Fisker, N.; Hedtjarn, M.;
ACKNOWLEDGMENTS
■
We appreciate financial support from the Idaho NSF EPSCoR,
the BANTech Center at the University of Idaho, and Award
No. GM088697 from the National Institute of General Medical
Sciences, National Institutes of Health. Scholarships from the
College of Graduate Studies (M.E.Ø.) and the National Science
Foundation under Award No. 0648202 and the Department of
Defense ASSURE (Awards to Stimulate and Support Under-
graduate Research Experiences) Program (D.J.R.) are appre-
ciated. We thank Dr. Alexander Blumenfeld (Department of
Chemistry), Dr. Gary Knerr (Department of Chemistry) and
Dr. Lee Deobald (EBI Murdock Mass Spectrometry Center,
University of Idaho) for NMR and mass spectrometric analyses.
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Article
Lindholm, M. W.; Rosenbohm, C.; Aarup, V.; Hansen, H. F.; Ørum,
H.; Hansen, J. B.; Koch, T. Nucleic Acids Res. 2010, 38, 7100−7111.
(d) Lanford, R. E.; Hildebrandt-Eriksen, E. S.; Petri, A.; Persson, R.;
Lindow, M.; Munk, M. E.; Kauppinen, S.; Ørum, H. Science 2010, 327,
198−201. (e) Obad, S.; Dos Santos, C. O.; Petri, A.; Heidenblad, M.;
Broom, O.; Ruse, C.; Fu, C.; Lindow, M.; Stenvang, J.; Straarup, E. M.;
Hansen, H. F.; Koch, T.; Pappin, D.; Hannon, G. J.; Kauppinen, S.
Nat. Genet. 2011, 43, 371−378.
(23) Kumar, P.; Østerggard, M. E.; Hrdlicka, P. J. Curr. Protocols
Nucleic Acid Chem. 2011, 44, 4.43.1−4.43.22.
(24) Terminal alkynes were obtained from commercial vendors,
synthesized via EDC-mediated coupling between functionalized
carboxylic acids and propargylamine (see Scheme S1, Supporting
Information) or prepared according to known literature protocols (see
the Experimental Section).
(25) (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett.
1975, 50, 4467−4470. (b) Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y.
Chem. Rev. 2003, 103, 1875−1916.
(26) Schrock, A. K.; Schuster, G. B. J. Am. Chem. Soc. 1984, 106,
5234−5240.
(27) Park, S. Y.; Yoon, J. H.; Hong, C. S.; Souane, R.; Kim, J. S.;
Mathews, S. E.; Vicens, J. J. Org. Chem. 2008, 73, 8212−8218.
(28) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596−2599.
(29) Bryld, T.; Lomholt, C. Nucleosides Nucleotides Nucleic Acids
2007, 26, 1645−1647.
(30) Mergny, J. L.; Lacroix, L. Oligonucleotides 2003, 13, 515−537.
(31) Gyi, J. I.; Gao, D.; Conn, G. L.; Trent, J. O.; Brown, T.; Lane, A.
N. Nucleic Acids Res. 2003, 31, 2683−2693.
(32) Demidov, V. V.; Frank-Kamenetskii, M. D. Trends Biochem. Sci.
2004, 29, 62−71.
(33) (a) Korshun, V. A.; Stetsenko, D. A.; Gait, M. J. J. Chem. Soc.,
Perkin Trans. 1 2002, 1092−1104. (b) Dohno, C.; Saito, I.
ChemBioChem 2005, 6, 1075−1081. (c) Kumar, T. S.; Madsen, A.
S.; Østergaard, M. E.; Sau, S. P.; Wengel, J.; Hrdlicka, P. J. J. Org.
Chem. 2009, 74, 1070−1081.
(34) Marin, V.; Hansen, H. F.; Koch, T. R.; Armitage, B. A. J. Biomol.
Struct. Dyn. 2004, 21, 841−850.
(35) (a) Mayer, E.; Valis, L.; Wagner, C.; Rist, M.; Amann, N.;
Wagenknecht, H.-A. ChemBioChem 2004, 5, 865−868. (b) Hwang, G.
T.; Seo, Y. J.; Kim, S. J.; Kim, B. H. Tetrahedron Lett. 2004, 45, 3543−
3546.
(36) (a) Dougherty, G.; Pilbrow, J. R. Int. J. Biochem. 1984, 16,
1179−1192. (b) Manoharan, M.; Tivel, K. L.; Zhao, M.; Nafisi, K.;
Netzel, T. L. J. Phys. Chem. 1995, 99, 17461−17472. (c) Seo, Y. J.;
Ryu, J. H.; Kim, B. H. Org. Lett. 2005, 7, 4931−4933.
(37) Østergaard, M. E.; Kumar, P.; Baral, B.; Guenther, D. C.;
Anderson, B. A.; Ytreberg, F. M.; Deobald, L.; Paszczynski, A. J.;
Sharma, P. K.; Hrdlicka, P. J. Chem. Eur. J. 2011, 17, 3157−3165.
(38) Seitz, O.; Schmuck, E. C.; Wennemers, H. Homogeneous DNA
Detection. In Highlights in Bioorganic Chemistry; Wiley-VCH:
Weinheim, Germany, 2004;pp 311−328.
(39) Quantum yields were not determined for Z-modified duplexes.
However, direct comparison with Z5d:DNA, which has a relative
fluorescence quantum yield of ∼16%,18j suggests that Z5:DNA has a
quantum yield of 20−30%.
(40) (a) Yamana, K.; Iwase, R.; Furutani, S.; Tsuchida, H.; Zako, H.;
Yamaoka, T.; Murakami, A. Nucleic Acids Res. 1999, 27, 2387−2392.
(b) Yamana, K.; Zako, H.; Asazuma, K.; Iwase, R.; Nakano, H.;
Murakami, A. Angew. Chem., Int. Ed. 2001, 40, 1104−1106. (c) Mahara,
A.; Iwase, R.; Sakamoto, T.; Yamana, K.; Yamaoka, T.; Murakami, A.
Angew. Chem., Int. Ed. 2002, 41, 3648−3650. (d) Hrdlicka, P. J.; Babu,
B. R.; Sørensen, M. D.; Harrit, N.; Wengel, J. J. Am. Chem. Soc. 2005,
127, 13293−13299. (e) Astakhova, I. V.; Korshun, V. A.; Wengel, J.
Chem. Eur. J. 2008, 14, 11010−11026. (f) Wang, G.; Bobkov, G. V.;
Mikhailov, S. N.; Schepers, G.; van Aerschot, A.; Rozenski, J.; van der
Auweraer, M.; Herdewijn, P.; Feyter, S. D. ChemBioChem 2009, 10,
1175−1185. (g) Østergaard, M. E.; Cheguru, P.; Papasani, M. R.; Hill,
R. A.; Hrdlicka, P. J. J. Am. Chem. Soc. 2010, 132, 14221−14228.
(h) Astakhova, I. V.; Lindegaard, D.; Korshun, V. A.; Wengel, J. Chem.
(14) For an updated clinical status of LNA, see: http://www.santaris.
com/product-pipeline.
(15) Wienholds, E.; Kloostermann, W. P.; Misk, W. P.; Alvarez-
Saavedra, E.; Berezikov, E.; Bruijn, E.; Horvitz, H. R.; Kauppinen, S.;
Plasterk, R. H. A. Nature 2005, 309, 310−311.
(16) For particularly interesting earlier examples see: (a) Sørensen,
M. D.; Kværnø, L.; Bryld, T.; Hakansson, A. E.; Verbeure, B.; Gaubert,
̊
G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 2164−2176.
(b) Sørensen, M. D.; Petersen, M.; Wengel, J. Chem. Commun. 2003,
2130−2131. (c) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.;
Tsutsumi, S.; Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg.
Med. Chem. 2003, 11, 2211−2226. (d) Fluiter, K.; Frieden, M.;
Vreijling, J.; Rosenbohm, C.; De Wissel, M. B.; Christensen, S. M.;
Koch, T.; Ørum, H.; Baas, F. ChemBioChem 2005, 6, 1104−1109.
(e) Albæk, N.; Petersen, M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731−
7740. (f) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.;
Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128,
15173−15187. (g) Abdur Rahman, S. M.; Seki, S.; Obika, S.;
Yoshikawa, H.; Miyashita, K.; Imanishi, T. J. Am. Chem. Soc. 2008,
130, 4886−4896. (h) Mitsuoka, Y.; Kodama, T.; Ohnishi, R.; Hari, Y.;
Imanishi, T.; Obika, S. Nucleic Acids Res. 2009, 37, 1225−1238.
(i) Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych, O.;
Chattopadyaya, J. J. Org. Chem. 2009, 74, 118−134. (j) Seth, P. P.;
Siwkowski, A.; Allerson, C. R.; Vasquez, G.; Lee, S.; Prakash, T. P.;
Wancewicz, E. V.; Witchell, D.; Swayze, E. E. J. Med. Chem. 2009, 52,
10−13.
(17) For reviews, see: (a) Luyten, I.; Herdewijn, P. Eur. J. Med. Chem.
1998, 33, 515−576. (b) Ahmadian, M.; Bergstrom, D. E. In Modified
Nucleotides in Biochemistry, Biotechnology and Medicine, 1st ed
.;Herdewijn, P., Ed.; Wiley-VCH: Weinheim, Germany, 2008; pp
251−276.
(18) For particularly interesting examples from the original research
literature, see: (a) Wagner, R. W.; Matteucci, M. D.; Lewis, J. G.;
Gutierrez, A. J.; Moulds, C.; Froehler, B. C. Science 1993, 260, 1510−
1513. (b) Hashimoto, H.; Nelson, M. G.; Switzer, C. J. Am. Chem. Soc.
1993, 115, 7128−7134. (c) Sagi, J.; Szemzo, A.; Ebinger, K.; Szabolcs,
A.; Sagi, G.; Ruff, E.; Otvos, L. Tetrahedron Lett. 1993, 34, 2191−2194.
(d) Ahmadian, M.; Zhang, P. M.; Bergstrom, D. E. Nucleic Acids Res.
1998, 26, 3127−3135. (e) Heystek, L. E.; Zhou, H. Q.; Dande, P.;
Gold, B. J. Am. Chem. Soc. 1998, 120, 12165−12166. (f) Kottysch, T.;
Ahlborn, C.; Brotzel, F.; Richert, C. Chem. Eur. J. 2004, 10, 4017−
4028. (g) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004,
126, 4820−4827. (h) Booth, J.; Brown, T.; Vadhia, S. J.; Lack, O.;
Cummins, W. J.; Trent, J. O.; Lane, A. N. Biochemistry 2005, 44,
4710−4719. (i) Skorobogatyi, M. V.; Malakhov, A. D.; Pchelintseva, A.
A.; Turban, A. A.; Bondarev, S. L.; Korshun, V. A. ChemBioChem 2006,
7, 810−816. (j) Østergaard, M. E.; Guenther, D. C.; Kumar, P.; Baral,
B.; Deobald, L.; Paszczynski, A. J.; Sharma, P. K.; Hrdlicka, P. J. Chem.
Commun. 2010, 4929−4931.
(19) (a) Barbaric, J.; Wagenknecht, H. A. Org. Biomol. Chem. 2006, 4,
2088−2090. (b) Kocalka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P.
ChemBioChem 2007, 8, 2106−2116. (c) Nguyen, T. N.; Brewer, A.;
Stulz, E. Angew. Chem., Int. Ed. 2009, 48, 1974−1977. (d) Kaura, M.;
Kumar, P.; Hrdlicka, P. J. Org. Biomol. Chem. 2012, 10, 8575−8578.
(20) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010, 110,
2579−2619.
(21) For a recent example, see: Andersen, N. K.; Anderson, B. A.;
Wengel, J.; Hrdlicka, P. J. J. Org. Chem. 2013, 78, 12690−12702.
(22) Østergaard, M. E.; Kumar, P.; Baral, B.; Raible, D. J.; Kumar, T.
S.; Anderson, B. A.; Guenther, D. C.; Deobald, L.; Paszczynski, A. J.;
Sharma, P. K.; Hrdlicka, P. J. ChemBioChem 2009, 10, 2740−2743.
Commun. 2010, 46, 8362−8364. (i) Forster, U.; Lommel, K.; Sauter,
̈
D.; Grunewald, C.; Engels, J. W.; Wachtveitl, J. ChemBioChem 2010,
̈
11, 664−672. (j) Mansawat, W.; Boonlua, C.; Siriwong, K.; Vilaivan, T.
Tetrahedron 2012, 68, 3988−3995.
(41) Shen, L.; Siwkowski, A.; Wancewicz, E. V.; Lesnik, E.; Butler,
M.; Witchell, D.; Vasquez, G.; Ross, B.; Acevedo, O.; Inamati, G.;
5060
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The Journal of Organic Chemistry
Article
Sasmor, H.; Manoharan, M.; Monia, B. P. Antisense Nucleic Acid Drug
Dev. 2003, 13, 129−142.
(42) Kumar, P.; Baral, B.; Anderson, B. A.; Guenther, D. C.;
Østergaard, M. E.; Sharma, P. K.; Hrdlicka, P. J. J. Org. Chem. 2014,
DOI: 10.1021/jo5006153.
(43) For structures of alkynes Ae−g, see Scheme S1 (Supporting
Information).
(44) Suenaga, M.; Kaneko, Y.; Kadokawa, J.; Nishikawa, T.; Mori, H.;
Tabata, M. Macromol. Biosci. 2006, 6, 1009−1018.
́ ́
(45) Godeau, G.; Barthelemy, P. Langmuir 2009, 25, 8447−8450.
(46) Ott, K.; Schmidt, K.; Kircher, B.; Schumacher, P.; Wiglenda, T.;
Gust, R. J. Med. Chem. 2005, 48, 622−629.
(47) Trybulski, E.; Zhang, J.; Kramss, R. H.; Mangano, R. M. J. Med.
Chem. 1993, 36, 3533−3541.
(48) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997,
62, 7512−7515.
(49) Qu, J.; Shiotsuki, M.; Sanda, F.; Masuda, T. Macromol. Chem.
Phys. 2007, 208, 823−832.
(50) Wenting, W.; Wanhua, W.; Shaomin, J.; Hunin, G.; Jiazhang, Z.
Eur. J. Inorg. Chem. 2010, 4470−4482.
(51) Andronova, V. L.; Skorobogtyi, M. V.; Manasova, E. V.; Berlin,
Y. A.; Korshun, V. A.; Galegov, G. A. Russ. J. Bioorg. Chem. 2003, 29,
262−266.
(52) Initial screening revealed that this activator results in higher
stepwise coupling yields than the normal 4,5-dicyanoimidazole
activator.
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